ES2798147T3 - Combination of two genetic elements to control the development of the floral type of a dicotyledonous plant, and use in detection and selection procedures - Google Patents

Abstract

Use of a combination of two genetic elements to control the development of the floral type of a dicotyledonous plant, said combination comprising, respectively: a) a first genetic control element (A / a) present in said dicotyledonous plant, in the form of a dominant allele (A) and one recessive allele (a), in which: - the dominant allele (A) consists of a nucleic acid (NA) that allows the expression of the ACS protein (aminocyclopropane-carboxylate synthase) of sequence SEQ ID No. 3 - the recessive allele (a) is distinguished from the dominant allele in that said recessive allele (a) is non-functional in said dicotyledonous plant, and b) a second control genetic element (G / g) present in said dicotyledonous plant, in form of a dominant allele (G) and a recessive allele (g), in which: - the dominant allele (G) consists of a nucleic acid (NG) that allows the expression of the protein CmWIP1 ("C. melo Zinc Finger Protein ") of sequence SEQ ID No. 12, - the recessive allele (g ) is distinguished from the dominant allele in that said recessive allele (g) is non-functional in said dicotyledonous plant, it being understood that at least the second control genetic element has been artificially introduced into said dicotyledonous plant.

Description

DESCRIPTION

Combination of two genetic elements to control the development of the floral type of a dicotyledonous plant, and use in detection and selection procedures

Field of the invention

The present invention relates to the field of the selection of plant varieties and, in particular, to the selection of the sexual type of plants. It refers to the genotypic detection of the sex of plants by analysis of the polymorphism of a gene A and a gene G, as well as means of using this detection in procedures for obtaining plants whose sexual phenotype is modified.

Previous technique

The realization of hybrid plants is of great interest in agronomy and agriculture. In fact, hybrid plants, thanks to the phenomenon of heterosis also called hybrid vigor, present a superiority in numerous characters with respect to the average of their two parents. This superiority can be used, for example, for a better vigor, a better yield, a greater adaptation to the environment in which the hybrid is cultivated and a great uniformity of the hybrids with respect to their parents. This hybrid vigor is all the more important the further the parents are genetically distant.

The creation of pure and stable lines, future progenitors of the hybrid, is an obligatory step for the creation of homogeneous and reproducible hybrid varieties that express the maximum of heterosis. Therefore, it is necessary to create pure and stable lines, then grow these lines to obtain hybrids.

The creation of pure lines implies the self-fertilization of a plant in such a way as to obtain plants that present the same genetic heritage set by the set of productivity, regularity and yield characters or even resistance to diseases, which are investigated.

To create pure lines it is therefore necessary to use plants whose sexual type allows self-fertilization, for example, hermaphrodite plants.

However, many of the dicotyledonous plants, and in particular the cucurbits, can be monoecious, andromonoean, gynoean, or hermaphroditic.

A first technique used to obtain pure lines consists of carrying out a chemical treatment of the plants, so as to obtain plants suitable for self-fertilization, for example, hermaphrodite plants. In the case of melon ( cucumis meto) for example, the spraying of ethylene synthesis inhibitors such as silver nitrate or silver thiosulfate implies the temporary appearance of stamens in female flowers (Rudich et al., 1969; Risser et al. ., 1979). In this way, the transformation of gynoecious plants into hermaphroditic plants is used for the maintenance of pure lines.

However, the production of pure lines by this method is limited by the cost of the chemical agents, the duration of their action and their cytotoxic effects. Furthermore, these agents may not be effective when it comes to making hybrids from plants whose flowering time is long, since the new flowers that appear after the treatment may not be affected by the chemical treatment.

Therefore, there is a need for a system that allows controlling the development of the floral type of a dicotyledonous plant and obtaining a plant of a particular floral type.

Furthermore, very numerous growths are necessary to obtain interesting hybrids from pure lines and, in each growth, the plants showing the most promising phenotype are retained.

During the growth of the pure lines between them, it is essential to be able to choose the direction of the growth carried out and avoid the self-pollination of the plants that would lead to plants that do not present the hybrid vigor sought.

Still in this case, due to the diversity of the sexual type of dicotyledonous plants, it is necessary to separate the male flowers and the female flowers of the same plant to avoid self-pollination.

In a first technique, carried out particularly for corn, it consists in using mechanical means to emasculate the plants. However, this technique proves to be extremely expensive since it requires the emasculation of each plant of which self-pollination is to be avoided for each growth carried out. Another technique consists in carrying out a chemical emasculation of the plants, blocking the formation of viable pollen. Thus, in the case of melon ( Cucumis meto), the treatment of monoecious plants by etrel (ethylene precursor) implies the temporary disappearance of male flowers.

These chemical agents, called gematocides, used to cause transient male sterility, have several drawbacks such as high cost or high toxicity, as already indicated above.

The mechanical or chemical control techniques of the floral type described above prove to be very expensive, insofar as very numerous growths are necessary to obtain hybrid plants that present the desired characteristics and that can be commercialized.

To facilitate the creation of pure lines and hybrids, therefore, there is also a need for a system that allows controlling the development of the floral type of a dicotyledonous plant and obtaining a plant of a specific floral type.

Another way to obtain plants capable of self-pollination useful for the creation of pure lines, or not capable of self-pollination, for the creation of hybrids could consist, respectively, of a selection of exclusively hermaphroditic individuals, or exclusively female, present in a species. However, this technique would prove extremely expensive as well, since it would require the cultivation of a very considerable number of plants, until such time as it is possible to determine the sexual type. This technique would also be random, since the mechanisms for determining the sex of the flowers depend particularly on environmental factors. According to yet another way, it has been sought to identify and characterize the genetic determinants that participate in the control of the floral type in melon. In the case of melon, the genetic control of sex determination is governed by two main genetic determinants, respectively (1) the andromonoic genetic determinant ( “Andromoeocious” or “a”) and (2) the genoecious genetic determinant ( “gynoecious” or "g"), each determinant having at least two alleles and whose combinations produce a great variety of sexual phenotypes. The international PCT application published under No. WO 2007/125264 describes the identification and characterization of the genetic determinant (a) which shows that it consists of a gene encoding an aminocyclopropane carboxylate synthase (ACS). Therefore, PCT application No. 2007/12564 provides detection and control means that allow selecting or generating dicotyledonous plants that possess the dominant allele (a) or the recessive allele (a). The genetic determinant (g) remains totally unknown. Furthermore, preliminary data suggested that the genetic determinant (g), of unknown nature and structure, could be located in a wide genomic region of more than 2.4 megabases delimited by markers called M8 and M30. Since it is commonly accepted that there is an average of 12 open reading frames ("ORFs") in 100 kilobases of plant genome, the genomic region bounded between markers M8 and M30 could contain approximately 300 open reading frames.

However, in the absence of characterization of the second gynoic genetic determinant (or "g"), it was not possible to make available means of selection or control of the development of the floral type that allow, for example, to discriminate or to generate a population of plants strictly females, since this phenotype is controlled exclusively by the genetic determinant (g). Likewise, the selection or obtaining of a population of exclusively hermaphroditic plants would only be possible after the identification and characterization of the genetic determinant (g).

Therefore, there is also a need for a method that allows the selection of dicotyledonous plants, for example hermaphrodites or females, without the need for cultivating them.

This procedure should make it possible to select plants, particularly useful for the realization of pure lines or hybrids, as has been specified previously.

Compendium of the invention

According to the invention, the genetic determinant (g) gynoic has been identified and characterized for the control of the floral development of a dicotyledonous plant that is an angiosperm and, more precisely, a plant of the cucurbit family.

The identification and characterization of the genetic determinant (g) gynoic allowed for the first time the development of a combination of the two genetic determinants (a) andromonoic and (g) gynoic that allow to fully control the development of the floral type of a plant dicotyledonous, whatever the sexual phenotype that is considered.

The invention therefore relates to a combination of two genetic elements (A / a) and (G / g) that makes it possible to control the development of the floral type of a dicotyledonous plant, in particular of a cucurbit such as melon.

It has already been shown in the state of the art that, physiologically, the two alleles (A) and (a) are distinguished by different proportions of enzymatic activity of a protein, aminocyclopropane-carboxylate synthase, also called ACS.

The inventors have now shown that physiologically, the two alleles (G) and (g) which have been identified and characterized according to the invention, are distinguished by different proportions of a new protein, the CmWIP1 protein.

The object of the invention is therefore the use of a combination of two genetic elements for the control of the development of the floral type of a dicotyledonous plant, said combination comprising, respectively: a) a first genetic control element (A / a) present in said dicotyledonous plant, in the form of a dominant allele (A) and a recessive allele (a), in which:

- the dominant allele (A) consists of a nucleic acid (NA) that allows the expression of the ACS protein (aminocyclopropane-carboxylate synthase) of sequence SEQ ID N ° 3,

- the recessive allele (a) is distinguished from the dominant allele in that said recessive allele (A) is non-functional in said dicotyledonous plant, and

b) a second genetic control element (G / g) present in said dicotyledonous plant, in the form of a dominant allele (G) and a recessive allele (g), in which:

- the dominant allele (G) consists of a nucleic acid (NG) that allows the expression of the CmWIPI protein of the sequence SEQ ID N ° l2

- the recessive allele (g) is distinguished from the dominant allele in that said recessive allele (g) is non-functional in said dicotyledonous plant,

it should be understood that at least the second genetic control element has been artificially introduced into said dicotyledonous plant.

In certain embodiments according to the invention, for the second control genetic element (G / g), the respective characteristics of the dominant allele (G) and the recessive allele (g) are the following:

- the dominant allele (G) consists of a nucleic acid (NG) comprising:

(i) a regulatory polynucleotide (PG) functional in a dicotyledonous plant and

(ii) a nucleic acid whose expression is regulated by the regulatory polynucleotide (PG), in which said nucleic acid encodes the CmWlPI protein of sequence SEQ ID N ° 12,

the recessive allele (g) is distinguished from the dominant allele (G) by a non-functional nucleic acid and comprises:

(i) a regulatory polynucleotide (Pg) whose nucleotide sequence comprises at least one mutation, an insertion or a deletion with respect to the nucleic acid of sequence SEQ ID N ° 13 leading said nucleic acid to a reduced or null expression of the CmWIP1 protein of sequence SEQ ID No. 12, or

(ii) a nucleic acid (Ng) that encodes a protein that differs from the CmWIP1 protein of sequence SEQ ID N ° 12 by the substitution, insertion or deletion of one or more amino acids and that does not possess the biological activity of said protein CmWIP1 in said dicotyledonous plant.

The applicant describes an ACS protein that encompasses the protein of sequence SEQ ID No. 3 or a protein that has at least 90% amino acid identity, preferably, at least 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98% or 99% identity in amino acids, with the protein of sequence SEQ ID N ° 3.

The applicant describes a CmWIP1 protein that encompasses the protein of sequence SEQ ID No. 12 or a protein that has at least 90% amino acid identity, preferably at least 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98% or 99% amino acid identity with the protein of sequence SEQ ID No. 12. The CmWIP1 protein can also encompass the protein of sequence SEQ ID No. 16 or a protein having at least one 90% amino acid identity, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity with the protein of sequence SEQ ID No. 16.

In certain embodiments according to the invention, for the second control genetic element (A / a), the respective characteristics of the dominant allele (A) and of the recessive allele (a) are the following:

- the dominant allele (A) consists of a nucleic acid (NA) comprising:

(i) a regulatory polynucleotide (PA) functional in a dicotyledonous plant, and

(ii) a nucleic acid whose expression is regulated by the regulatory polynucleotide (PA), said nucleic acid encoding the ACS protein (aminocyclopropane carboxylate synthase) of sequence SEQ ID N ° 3,

- the recessive allele (a) is distinguished from the dominant allele (A) by a non-functional nucleic acid and comprises:

(i) a regulatory polynucleotide (Pa) whose nucleotide sequence comprises an insertion, a substitution or a deletion with respect to the nucleic acid that goes from nucleotide 1 to nucleotide 5907 of the sequence SEQ ID N ° 1 and that leads to a reduced or no expression of the ACS protein of sequence SEQ ID N ° 3, or

(ii) a nucleic acid (Na) that encodes a protein that differs from the ACS protein of sequence SEQ ID N ° 3 by the substitution, insertion or deletion of one or more amino acids and that does not possess the biological activity of said protein ACS in said dicotyledonous plant.

The application also describes regulatory polynucleotides (PG) and (Pg) as such.

The invention also relates to methods for obtaining a transformed plant whose sexual phenotype has been modified, as well as the parts of this plant, particularly its seeds.

Another subject of the invention is the CmWIP1 protein as defined in more detail below, or a fragment of this protein, as well as antibodies directed against the CmWIP1 protein.

The invention also deals with methods for detecting the presence of alleles (A), (a), (G) and (g) in a sample.

Description of the figures

Figure 1 illustrates the positional cloning of the g site.

Figure 1A illustrates the physical and genetic maps of site (g) on chromosome 4. The site is bordered by two markers M261 and M335. The dashed lines indicate the position of these genetic markers in the different BAC clones.

Figure 1B illustrates the representation of 8 open reading frames or ORFs (long arrow) found in the BAC 102, with their orientation prediction. The triangular reference represents the insertion of the DNA transposon called Gyno-hAT whose sequence is described in SEQ ID No. 14. The insertion of this transposon is what induces the methylation of the promoter of the CmWIP1 gene and leads to its inactivation.

Figure 1C is a high resolution map of two critical recombination events in the vicinity of the g site. SNP polymorphisms are indicated and recombinants P63.2 and 87.94 determined a final region of 1.4 kb.

Figure 2 illustrates the results of McrBr endonuclease-sensitive semi-quantitative PCR amplification on transposon DNA inserted at the g site.

The sense primer is located in the sequence of the transposon and the antisense primer is located in the genomic sequence bordering the transposon. PCR amplification without endonuclease digestion of the Gynadou genomic DNA resulted in strong amplification, meaning the presence of the transposon there. PCR amplification with McrBC endonuclease predigestion shows no amplification, indicating a very high level of transposon methylation. No amplification was obtained for PI124112 whatever the support, because the transposon was not inserted in the G site.

Figure 3 illustrates the g-site methylation analysis.

Figure 3A illustrates McrBR-sensitive semi-quantitative PCR amplification over three open reading frames or ORFs, closest to the locus for the monoecious (G-) PI1214112 genotype and the Gynadou (gg) gynoic genotype. The absence of pre-digestion amplification with McrBC indicates the presence of DNA methylation. The oligonucleotides were designed in order to generate an amplicon that borders the predicted transcription start site for each OFR, which includes a part of the promoter and a part of the first exon.

Figure 3B illustrates DNA methylation and the structure of the CmWIP1 gene. The black arrows represent the start site in the transcription determined by the "5'RACE" technique, the black compartments represent two exons, the symbol after the second exon represents the end of the "3'UTR" sequence, determined by the technique. of "3'RACE".

The insertion of the transposon is symbolized in the area over the 3 'end of the gene. DNA methylation in the entire CmWIP1 sequence was determined by quantitative PCR amplification sensitive to McrBC. Each value corresponds to the mean of at least three plants, each PCR reaction being performed in triplicate.

Figure 3C illustrates the methylation analysis of cytosines by bisulfite sequencing. Two amplicons corresponding to the highly methylated part of the promoter were amplified after bisulfite treatment. The percentage of methylated cytosines is indicated by vertical bars.

Figure 4 illustrates the g-site methylation analysis for different genetic sources of C. melo. W1998, Bulgarie14, Paul and Gynadou are homozygous for the g allele. PI161375 Vedrantais and PI124112 are homozygous for the G allele.

Figure 4A illustrates the experiment by which the insertion of the transposon at the g-site was selected by PCR amplification in the different genetic sources. The sense primer was located in the transposon sequence and the antisense primer was located in the genomic sequence bordering the transposon in order to verify the presence of the insert (upper line), in which the two primers bordering the Insertion site were used in order to verify the absence of the transposon (bottom line).

Figure 4A represents the results of McrBr-sensitive semi-quantitative PCR amplification in the CmWIP1 gene in different genetic sources. The absence of amplification after a digestion with McrBc indicates the presence of DNA methylation.

Figure 5 illustrates the analysis of the expression profile of CmWIP1 messenger RNA. The expression levels of CmWIP1 were analyzed by quantitative PCR. Each value corresponds to the average of at least three plants. The expression levels of CmWIP1 were normalized to the expression levels of a unit gene, the actin gene. Figure 6 illustrates the results of the analysis of the expression levels of CmWIP1 analyzed by quantitative PCR in a set of flower buds up to stage 6. Each value corresponds to the mean of at least three plants. The expression levels of CmWIP1 were normalized to the expression levels of a ubiquitous gene, the actin gene.

Figure 7 illustrates the observation of floral phenotypes identified by the TILLING technique. The main stem flowers of the S306F mutant as well as that of the P193L mutant were compared to a male flower and a wild type female flower of monoecious parent. S306F mutant flowers such as that of the P193L mutant clearly show fourth spiral ovary development. The L77F mutant is a weak mutant. Ow: ovary; St: stamen.

Figure 8A presents an alignment of an ACS gene messenger RNA fragment from a plant having phenotype (A) [top line] and from a plant having phenotype (a) [bottom line] and illustrates a point mutation of a nucleotide that differentiates (A) and (a).

Figure 8B represents an alignment of the nucleic acid-encoded amino acid sequences of Figure 8A, comprising a plant having phenotype (A) [top line] and a plant having phenotype (a) [second line] and illustrates a point mutation of an amino acid that differentiates (A) and (a).

Figure 9 depicts micrographs of transgenic Arabidopsis thaliana plants carrying the A or a melon allele. Figures 9A and 9B show that the siliques of plants transformed with the A allele (Fig. 9A-Cm-A and Fig. 9B) are shorter than the siliques of wild plants (Col-0) and plants transformed with allele (a).

Detailed description of the invention

According to the present invention, the genetic determinant (G / g) that controls, in combination with the genetic determinant (A / a) previously described in the state of the art, the development of the floral type in cucurbits has been identified and characterized.

The identification and characterization of the genetic determinant (G / g) provides for the first time to a person skilled in the art the possibility of selecting or generating dicotyledonous plants that have a desired sexual phenotype, in particular cucurbits such as melon that have the desired sexual phenotype .

It should be remembered that the allele (A) controls the andromonoic character of the plants and the allele (G) controls the gynoic character of the plants, as illustrated in Table 1 below.

Table 1

Table 1 illustrates the correspondence between the genotype and the sexual phenotype of the flowers of dicot plants. The inventors have now shown that, physiologically, the two alleles (G) and (g), which have been identified and characterized according to the invention, are distinguished in the different proportions of a new protein, the CmWIP1 protein. By making sequence comparisons with known proteins, the inventors have shown that the novel CmWIP1 protein could be classified in the family of WIP-like zinc finger proteins found in a wide variety of plants, including dicots, monocots, gymnosperms. and the mosses.

From a genetic point of view, the inventors have shown that allele (g) is distinguished from allele (G) in the reduced level of CmWIP1 protein in the plant, compared to a plant that carries an allele (G) or by the production of a protein CmWIP1 mutated with respect to the protein CmWIP1 produced by a plant that carries a (G) allele.

The inventors have also shown that allele (G) is dominant over allele (g).

As previously specified, it had already been shown in the state of the art that, physiologically, the two alleles (A) and (a) differ in the different proportions of enzymatic activity of a protein, aminocyclopropane-carboxylate synthase also called ACS, which is a protein involved in the synthesis of ethylene.

However, different studies have shown that cucurbit flower biology genes encode proteins involved in the ethylene biosynthesis or regulation pathway (Kamachi et al., 1997; Kahana et al., 2000). It was also previously shown that allele (a) is physiologically distinguished from allele (A) by the reduced level of enzyme activity of protein to Cs in the plant, compared to a plant carrying an allele (A).

It was also shown that the allele (A) is expressed in the primordia of the carpels and it is this expression that blocks the development of the stamens. The absence of expression of the A allele or the expression of a mutated form of the protein from TILLING selections, for example, or of the protein from the allele carrying the A57V mutation does not block the development of stamens. Finally, it was also shown that allele (A) is dominant over allele (a).

Although theoretical linkages are not desired, the inventors consider that developmental control syntheses of the floral type by a combination of alleles of the (G / g) and (A / a) gene can be generalized to the dicot family, including the Cucurbit family.

Therefore, the applicant describes a combination of two genetic elements and their use for the control of the development of the floral type of a dicotyledonous plant, said combination respectively comprising:

a) a first genetic control element (A / a) present in said dicotyledonous plant, in the form of a dominant allele (A) and a recessive allele (a), in which:

- the dominant allele (A) consists of a nucleic acid (NA) that allows the expression of the ACS protein (aminocyclopropane-carboxylate synthase), preferably the ACS protein of sequence SEQ ID N ° 3

- the recessive allele (a) is distinguished from the dominant allele in a non-functional nucleic acid (NA) in said dicotyledonous plant and

b) a second genetic control element (G / g) present in said dicotyledonous plant, in the form of a dominant allele (G) and a recessive allele (g), in which:

- the dominant allele (G) consists of a nucleic acid (NG) that allows the expression of the CmWIP1 protein, preferably the CmWIP1 protein of sequence SEQ ID N ° 12,

- the recessive allele (g) is distinguished from the dominant allele in a non-functional nucleic acid (NG) in said dicotyledonous plant,

it should be understood that at least the second genetic control element has been artificially introduced into said dicotyledonous plant.

The non-functional nucleic acid (NA) that characterizes the allele (a) encompasses (i) a nucleic acid that encodes a protein other than the protein of SEQ ID No. 3, comprising the mutated protein A57V, (ii) or any other Protein form mutated with respect to the protein of sequence SEQ ID No. 3 and leading to an inactive enzyme, (ii) or even other non-functional ACS (iv) or even an unexpressed allele.

The use of a combination of two genetic elements according to claim 1, characterized in that:

- the dominant allele (A), consisting of a nucleic acid (NA), comprises:

(i) a regulatory polynucleotide (PA) functional in a dicotyledonous plant, and

(ii) a nucleic acid whose expression is regulated by the regulatory polynucleotide (PA), said nucleic acid encoding the ACS protein (aminocyclopropane carboxylate synthase) of sequence SEQ ID N ° 3,

- the non-functional recessive allele (a) is distinguished from the dominant allele in a non-functional nucleic acid and comprises: (i) a regulatory polynucleotide (Pa) whose nucleotide sequence comprises an insertion, a substitution or a deletion with respect to the nucleic acid ranging from nucleotide 1 to nucleotide 5907 of the sequence SEQ ID No. 1 and leading to a reduced or no expression of the ACS protein of the sequence SEQ ID No. 3,

(ii) a nucleic acid (Na) that encodes a protein that differs from the ACS protein of sequence SEQ ID N ° 3 in the substitution, insertion or deletion of one or more amino acids and that does not possess the biological activity of said protein ACS in said dicotyledonous plant, and

- the dominant allele (G), consisting of a nucleic acid (NG), comprises:

(i) a regulatory polynucleotide (PG) functional in a dicotyledonous plant, and

(ii) a nucleic acid whose expression is regulated by the regulatory polynucleotide (PG), said nucleic acid encoding the expression of the CmWIP1 protein ("C. melo Zinc Finger Protein") of sequence SEQ ID N ° 12,

- the non-functional recessive allele (g) is distinguished from the dominant allele (G) in a non-functional nucleic acid and comprises:

(i) a regulatory polynucleotide (Pg) whose nucleotide sequence comprises at least one mutation, an insertion or a deletion with respect to the nucleic acid of sequence SEQ ID N ° 13, said nucleic acid leading to a reduced or null expression of the protein CmWIP1 of sequence SEQ ID No. 12 or

(ii) a nucleic acid (Ng) that encodes a protein that differs from the CmWIP1 protein of sequence SEQ ID N ° 12 in the substitution, insertion or deletion of one or more amino acids and that does not possess the biological activity of said protein CmWIP1 in said dicotyledonous plant.

The combination of the two genetic elements described above makes it possible to control and / or modify the sex of the flowers of dicotyledonous plants and, therefore, is very advantageous compared to mechanical, often expensive, or chemical control systems, often toxic, used in the prior art.

By "allele" is meant, in the sense of the present invention, one of the forms of a gene that occupies a site or place on a pair of homologous chromosomes. Alleles of a gene refer to the same genetic trait, but can determine different phenotypes.

A dominant allele is an allele whose level of phenotypic expression is much higher than that of the homologous allele (called recessive). The domain can be complete or partial.

A recessive allele is an allele that is only expressed in the phenotype when the plant receives identical alleles from each of its two parents. In contrast, the expression of the recessive allele is masked if the dominant homologous allele is present.

Therefore, the combination defined above exists in the form of different states, each corresponding to a phenotype.

When the first genetic control element (A / a) present in a dicotyledonous plant is in the form of an allele (A), the plant has a monoecious or gynoic phenotype.

When the first genetic control element (A / a) present in a plant is in the form of an allele (aa), the plant is hermaphroditic or andromonoic phenotype.

When the second genetic control element (G / g) present in a dicotyledonous plant is in the form of an allele (G), the plant has a monoecious or andromonoic phenotype.

When the second genetic control element (G / g) present in a plant is in the form of an allele (gg), the plant is of the hermaphroditic or gynoic phenotype.

The correspondence between alleles and phenotypes is summarized in Table 1.

In certain embodiments of the combination according to the invention, for the second control genetic element (G / g), the respective characteristics of the dominant allele (G) and the recessive allele (g) are the following:

- the dominant allele (G) consists of a nucleic acid (NG) comprising:

(i) a regulatory polynucleotide (PG) functional in a dicotyledonous plant, and

(ii) a nucleic acid whose expression is regulated by the regulatory polynucleotide (PG), said nucleic acid encoding the CmWIPI protein ( "C. melo Zinc Finger Protein") of sequence SEQ ID No. 12,

- the recessive allele (g) is distinguished from the dominant allele (G) in:

(i) a nucleic acid (NG) not present in the plant, or

(ii) a non-functional regulatory polynucleotide (Pg) in a dicotyledonous plant, or

(iii) a non-functional nucleic acid (Ng) for the expression of an active CmWIP1 protein.

In certain embodiments of the combination according to the invention, for the first control genetic element (A / a), the respective characteristics of the dominant allele (A) and the recessive allele (a) are the following:

- the dominant allele (A) consists of a nucleic acid (NA), comprising:

(i) a regulatory polynucleotide (PA) functional in a dicotyledonous plant, and

(ii) a nucleic acid whose expression is regulated by the regulatory polynucleotide (PA), said nucleic acid encoding the ACS protein (aminocyclopropane carboxylate synthase) of sequence SEQ ID N ° 3,

- the recessive allele (a) is distinguished from the dominant allele (A) in:

(i) a nucleic acid (NA) not present in the plant, or

(ii) a non-functional regulatory polynucleotide (Pa) in a dicotyledonous plant, or

(iii) a non-functional nucleic acid (Na) for the expression of an active ACS protein.

The following of the description sets forth preferred variants or embodiments of the first and second genetic control elements that form part of the control system object of the invention.

Control genetic element G / g, in the form of the dominant allele (G), in the combination of two genetic elements according to the invention.

In general, the control genetic element G / g, present in a plant as a dominant allele (G), allows to obtain a higher level of CmWIP1 protein, with respect to the level observed when the allele (G) is not present. in said plant.

In the description that follows, a "high level" of CmWIP1 protein is considered to correspond to the mean level of CmWIP1 protein measured in a plant that comprises the dominant G allele without its genome and a "low level" of CmWIP1 protein corresponds to the mean level of CmWIP1 protein observed in a plant that does not comprise the dominant allele (G) in its genome.

The dominant allele (G) consists of a nucleic acid (NG) comprising:

(i) a regulatory polynucleotide (PG) functional in a dicotyledonous plant, and

(ii) a nucleic acid whose expression is regulated by the regulatory polynucleotide (PG), said acid encoding nucleic acid the CmWIPI protein of sequence SEQ ID No. 12.

- Functional regulatory polynucleotide ( PG)

A functional promoter or regulatory polynucleotide (PG) according to the invention consists of a nucleic acid that allows the expression of the CmWIPI protein of sequence SEQ ID No. 12 in dicotyledonous plants.

An example mode of this promoter comprises or consists of the nucleic acid of sequence SEQ ID No. 13, which is located from nucleotide at position 1 to nucleotide at position 2999 of the nucleic acid of the CmWIP1 gene of sequence SEQ ID No. 10 .

Therefore, in the embodiments of the combination of the two genetic elements of the invention in which the second genetic element consists of the G allele, the regulatory polynucleotide (PG) can consist of a polynucleotide comprising or consisting of ( i) a nucleotide sequence ranging from nucleotide 1 to nucleotide 2999 of sequence SEQ ID No. 10 or (ii) a sequence that has at least 90% nucleotide identity with sequence 1-2999 of SEQ ID No. 10 and which is functional or (iii) a fragment of sequences (i) and (ii) above and which is functional.

Another subject of the invention is the regulatory polynucleotide (PG) as such, defined above, as well as fragments of this nucleic acid, as will be described in more detail in the part entitled "nucleic acids according to the" invention.

A functional regulatory polynucleotide (PG) according to the invention may also consist of a known promoter to digest the expression of nucleic acid sequences encoding the CmWIP1 protein in a constitutive or tissue-specific manner.

A functional regulatory polynucleotide (PG) according to the invention can therefore be chosen from promoters of specific tissues such as those of the genes of the family of "MADS box" of class ABCD and E, as described by Theipen et al., 2001 or any other promoter of homeotic genes.

A functional regulatory polynucleotide (PG) according to the invention can be chosen from:

- the 35S promoter of the cauliflower mosaic virus or the 19S promoter or, advantageously, the double constitutive 35S promoter (pd35S) described in the article by Kay et al., 1987;

- the ri3 actin promoter followed by the ri3 actin intron (pAR-IAR) contained in the plasmid pAct1F4 described by Mc Elroy et al., 1991;

- the constitutive EF-1a promoter of the gene encoding the plant elongation factor described in PCT application No. WO 90/02172 or even in the article by AXELOS et al. (1989);

- the chimeric superpromotor PSP (NI et al., 1995) constituted by the fusion of three copies of the element of transcriptional activity of the promoter of the octopine synthase gene of Agrobacterium tumefaciens and of the activating element of the transcription of the promoter of the gene of the Agrobacterium tumefaciens mannopine synthase ;

- the sunflower ubiquitin promoter (BINET et al., 1991);

- the ubiquitin 1 promoter from corn (CHRISTENSEN et al., 1996).

A functional regulatory polynucleotide (PG) according to the invention may also consist of an inducible promoter. The object of the invention is therefore a combination of two genetic elements for the control of the floral type of a dicotyledonous plant, as defined above, in which the regulatory polynucleotide (PG) is sensitive to the action of an inducing signal and , preferably, wherein the regulatory polynucleotide (PG) is an inducible transcriptional or translational activator polynucleotide.

When the transcriptional or translational activating regulatory polynucleotide is sensitive, directly or indirectly, to the action of an activating inducer signal, it is an "inducible activator" polynucleotide in the context of the invention.

According to the invention, an "inducible activator" regulatory polynucleotide is a regulatory sequence that is only activated in the presence of an external signal. This external signal can be the binding of a transcription factor, the binding of a transcription factor can be induced under the effect of the activating inducing signal to which the regulatory polynucleotide is sensitive directly or indirectly.

When this nucleic acid construct is used in a cellular host, the expression of the polynucleotide encoding the CmWIPI protein according to the invention can be induced by contacting the transformed cell host with the activating inducer signal to which the regulatory polynucleotide is directly or indirectly responsive.

When the absence of expression of the polynucleotide encoding a CmWIP1 polypeptide is sought in this transformed cell host, it is then sufficient to eliminate or suppress the presence of the activating inducing signal to which the transcriptional or translational regulatory polynucleotide is sensitive.

The person skilled in the art will be able to draw on their general technical knowledge in the field of regulatory polynucleotides, in particular those active in plants, to define the constructions that respond to the definition of the above embodiment.

The regulatory sequence capable of controlling the nucleic acid encoding a CmWIP1 protein can be a regulatory sequence inducible by a particular metabolite, such as:

- a glucocorticoid-inducible regulatory sequence as described by AOYAMA et al. (1997) or that described by McNELLYS et al. (1998);

- an ethanol-inducible regulatory sequence, as described by SALTER et al. (1998) or even the one described by CADDICK et al. (1998);

- a regulatory sequence inducible by tetracycline such as that marketed by the company CLONTECH;

- a promoter sequence inducible by a pathogen or by a metabolite produced by a pathogen;

- a regulatory sequence of genes of the PR type, inducible by salicylic acid or BTH or Aliette (Gorlach et al., 1996, Molina et al., 1998);

- a regulatory sequence of the Ecdysone receptor type (Martinez et al., 1999) inducible by tebufenozide (product reference RH5992, marketed by the company ROHM & HAAS), for example, belonging to the group of dibenzoylhydrazine.

- Nucleic acids encoding the CmWIPI protein

Preferably, the nucleic acid encoding the CmWIP1 protein comprises, at the 5 'end towards the 3' end, at least:

(i) a sequence that has at least 95% identity with the polynucleotide ranging from nucleotide 3000 to nucleotide 3617 of SEQ ID No. 10, and

(ii) a sequence that has at least 95% identity with the polynucleotide ranging from nucleotide 5458 to nucleotide 5901 of SEQ ID No. 10.

In other embodiments, the nucleic acid encoding the CmWIP1 protein comprises the nucleotide sequence SEQ ID No. 11.

Genetic control element G / g in the form of a recessive allele (g) of the system according to the invention

In general, the control genetic element (G / g) in the form of a recessive allele (g), when it is present in a plant that does not have the dominant allele (G) in its genome, does not allow to obtain a protein level CmWIP1 as high as that obtained when the allele (G) is present.

Therefore, allele (g) can be defined as any alteration of the genotype corresponding to allele (G), which does not allow obtaining a level of CmWIP1 as high as allele (G).

The recessive allele (g) is distinguished from the dominant allele (G) in:

(i) a nucleic acid (NG) not present in the plant, or

(ii) a non-functional regulatory polynucleotide (Pg) in a dicotyledonous plant, or

(iii) a non-functional nucleic acid (Ng) for the expression of a non-active CmWIP1 protein.

As an example, an embodiment of a G / g control element in the form of a recessive allele (g) is illustrated by the nucleic acid sequence SEQ ID No. 15, which is an allele of the sequence encoding CmWIP1 in which this present a transposon nucleic acid called "Gyno-hAT". In the nucleic acid of sequence SEQ ID No. 15, the Gyno-hAT transposon is located from nucleotide at position 7167 to nucleotide at position 15412 of sequence SeQ ID No. 15.

In the nucleic acid of sequence SEQ ID No. 14, the Gyno-hAT transposon is located from nucleotide at position 10 to nucleotide at position 8246 of sequence SEQ ID No. 14.

- Non-functional regulatory polynucleotide ( Pg)

A non-functional promoter or regulatory polynucleotide (Pg) according to the invention is a nucleic acid that:

(i) does not allow the expression of the CmWIP1 protein of sequence SEQ ID N ° 12 in a host cell, or (ii) allows the expression of this protein at a low level compared to the level observed with the regulatory polynucleotide (PG) , or

(iii) it allows the expression of the CmWIP1 protein during the life of the plant, for a shorter period, compared to that observed with the regulatory polynucleotide (PG).

In certain embodiments of the non-functional regulatory polynucleotide (Pg), said polynucleotide (Pg) is methylated. For example, the regulatory polynucleotide (Pg) may consist of a regulatory polynucleotide (PG) that occurs as a methylated nucleic acid.

By "methylated nucleic acid" or "methylated regulatory polynucleotide" is meant according to the invention the corresponding nucleic acid whose ratio (number of methylated bases) / (number of unmethylated bases) is at least 5/1. Therefore, according to the invention, a methylated nucleic acid encompasses a nucleic acid that has a ratio (number of methylated bases) / (number of unmethylated bases) of at least 6/1, 7/1, 8/1, 9 / 1, 10/1, 11/1, 12/1, 13/1, 14/1, 15/1, 16/1, 17/1, 18/1 and 20/1.

Determination of the ratio (number of methylated bases) / (number of unmethylated bases) can be easily carried out by one skilled in the art by any known technique. The person skilled in the art can use, in particular, the bisulfite sequencing method, as described in the examples of the present description. To compare the expression level of various promoters, a simple technique, known to one of ordinary skill in the art, is to place a selection marker gene under the control of the promoters to be tested. A selection marker gene can be, for example, the BASTA herbicide resistance gene, well known to one of skill in the art.

Another technique may consist of measuring the level of the CmWIP1 protein obtained when the sequence encoding this protein is under the control of different promoters, using antibodies directed to the encounter of this protein, and the procedures described in the "polypeptides according to the invention" part.

As already shown in the examples, an illustration of a non-functional promoter (Pg) consists of a promoter that has a nucleotide sequence equal to the nucleotide sequence of a functional promoter (PG) but that occurs in cellulo or in I live in a non-functional methylated form. In a specific embodiment illustrated in the examples, the methylated state of the promoter (Pg) is caused by the presence of an equivalent element (TE) located at a distance less than 1 kilobase from the 3 'end of the CmWIP1 gene.

A non-functional polynucleotide (Pg) can also be any polynucleotide derived from the polynucleotide (PG) as defined above, the nucleotide sequence of which comprises an insertion, a substitution or a deletion of one or more nucleotides, with respect to the nucleotide sequence of the regulatory polynucleotide.

Therefore, the object of the invention is also a nucleic acid that comprises a nucleotide sequence that carries at least one alteration chosen from a mutation, an insertion or a deletion, with respect to the nucleic acid that goes from nucleotide 1 to nucleotide 2999 of the sequence SEQ ID N ° 10, said altered nucleic acid leading to a reduced expression of the CmWIP1 protein, when it controls the expression of said protein, with respect to the expression of the CmWIP1 protein controlled by the nucleic acid that goes from the nucleotide 1 to nucleotide 2999 of the sequence SEQ ID No. 10.

Another subject of the invention is the regulatory polynucleotide (Pg) as such, as defined above. Another subject of the invention is a nucleic acid comprising a regulatory polynucleotide (Pg) and a nucleic acid encoding the CmWIP1 protein of SEQ ID N ° 12.

The invention also has as its object a combination of two genetic elements for the control of the development of the floral type of a dicotyledonous plant as defined in general in the present invention, in which the Regulatory polynucleotide (Pg) is sensitive to the action of an inducing signal and, preferably, where the regulatory polynucleotide (Pg) is an inducible transcriptional or translational repressor polynucleotide.

By "repressor" regulatory polynucleotide is meant, according to the invention, a regulatory sequence whose constitutive activity can be blocked by an external signal. This external signal may be the absence of binding of a transcription factor recognized by the repressor regulatory polynucleotide. The absence of binding of the transcription factor can be induced under the effect of the repressor inducing signal to which the repressor regulatory polynucleotide is sensitive.

According to this first particular embodiment, the expression of the sequence coding for a CmWIP1 protein is constitutive in the chosen cell host, in the absence of the repressor inducing signal to which the repressor regulatory polynucleotide is directly or indirectly sensitive.

The contact of the cell host with the repressor inducing signal has the effect, thanks to a direct or indirect action on the repressor regulatory polynucleotide, to inhibit and / or block the expression of the polynucleotide encoding the CmWIP1 protein.

In order to carry out the DNA constructions according to the invention comprising a repressor regulatory polynucleotide, the person skilled in the art will be able to draw on his general technical knowledge in the field of gene expression in plants.

A procedure for obtaining a transformed plant, using this type of regulatory polynucleotide, is described in the part entitled "Procedures for obtaining a transformed plant according to the invention".

- Nucleic acid ( Ng) not functional for the expression of an active CmWIPI protein

A nucleic acid (Ng) encompasses nucleic acids that comprise at least a part of a sequence that encodes an active CmWIP1 protein but does not allow, when placed under the control of a functional regulatory polynucleotide in dicot plant cells, the production of said plants of an active CmWIP1 protein.

A nucleic acid (Ng) essentially encompasses nucleic acids (NG) in which one or more mutations are present in at least one intron or exon, each mutation being chosen from (i) the substitution of one nucleotide or more than one nucleotide, (ii) the deletion of a nucleotide or of at least two consecutive nucleotides and (iii) the deletion of a nucleotide or of at least two consecutive nucleotides, with respect to the nucleic acid (NG) of reference. A nucleic acid (Ng) encompasses in particular nucleic acids that encode a non-active CmWIP1 protein.

By nucleic acid encoding a non-active CmWIP1 protein is meant, in the sense of the present invention, a nucleic acid encoding a protein that differs from the CmWIP1 protein of sequence SEQ ID No. 12 in substitution, deletion or insertion of one or more amino acids, and that does not possess the biological activity of the CmWIP1 protein of sequence SEQ ID N ° 12.

Also included is a nucleic acid that encodes a protein that differs from the CmWIP1 protein of sequence SEQ ID No. 16 in the substitution, deletion or insertion of one or more amino acids and that does not possess the biological activity of the CmWIP1 protein of sequence SEQ ID No. 16.

In particular, this non-active CmWIP1 protein, when it is expressed in a plant that does not express active CmWIP1 protein, in particular CmWIP1 protein of sequence s Eq ID No. 12, or of CmWIP1 protein of sequence SEQ ID No. 16, induces, respectively :

- a hermaphroditic plant phenotype in combination with the presence in homozygous form of alleles (a / a) in said plant,

- a phenotype of a female plant in combination with (i) the presence in the homozygous form of alleles (A / A) in said plant or in combination with (ii) the presence in the heterozygous form of alleles (A / a) in said plant.

It has been shown in the examples that plants possessing the allele (g) of the genetic element (G / g) were obtained with nucleic acids encoding an inactive CmWIP1 protein. In particular, it has been shown in the examples that plants that possess the allele (g) of the genetic element (G / g) were obtained with nucleic acids that encode a CmWIP1 protein that has a substitution of a single nucleotide in relation to the nucleotide sequence. SEQ ID No. 11 encoding the CmWIP1 protein of sequence SEQ ID No. 12.

For illustrative purposes, the examples show plants that possess the (g) allele and whose corresponding nucleic acid encodes a mutated CmWIP1 protein that has a substitution of an acid chosen from L77F, P193L and S306F, according to the amino acid numbering used for SEQ ID No. 12.

The genetic control element A / a, in the form of a dominant allele (A) or in the form of a dominant allele (a) has already been described in French patent application No. FR 2900415 and in PCT application No. WO 2007 / 125264.

However, since the genetic control element (A / a) is an important element of the combination of two genetic elements for the control of the development of the flower type according to the invention, its main characteristics are described again below.

Genetic control element A / a, in the form of the dominant allele (A) of the combination according to the invention

In general, the control genetic element A / a, present in a plant in the form of the dominant allele (A), makes it possible to obtain a higher level of active ACS protein, with respect to the level observed when the allele (A) does not is present in said plant.

In the description that follows, it is considered that a "high level" of ACS protein corresponds to the average level of ACS measured in a plant that comprises the dominant allele (A) in its genome and that a "low level" of ACS protein corresponds to the average level of active ACS protein observed in a plant that does not comprise the dominant allele (A) in its genome. A low level of ACS protein encompasses a null level of active ACS protein, for example, in the case of non-active a Cs expression, including the product of the allele a carrying the A57V mutation.

The dominant allele (A) consists of a nucleic acid (NA) comprising:

(i) a regulatory polynucleotide (PA) functional in a dicotyledonous plant; and

(ii) a nucleic acid whose expression is regulated by the regulatory polynucleotide (PA), said nucleic acid encoding the ACS protein of sequence SEQ ID N ° 3.

- Functional regulatory polynucleotide ( PA)

A functional promoter or regulatory polynucleotide (PA) according to the invention consists of a nucleic acid that allows the expression of the ACS protein of sequence SEQ ID No. 3 in dicotyledonous plants.

As an example, this promoter comprises a nucleotide sequence ranging from nucleotide 1 to nucleotide 5906 of the sequence S ^and Q ID No. 1.

Therefore, in the embodiments of the combination of two genetic elements of the invention in which the first genetic element consists of the A allele, the regulatory polynucleotide (PA) can consist of a polynucleotide that comprises or consists of (i) a nucleotide sequence ranging from nucleotide 1 to nucleotide 5906 of the sequence SEQ ID No. 1 or (ii) a sequence that has at least 90% nucleotide identity with the sequence 1-5906 of SEQ ID No. 1 and that it is functionalized or (iii) a fragment of sequences (i) and (ii) above that is functional.

Therefore, in certain embodiments of the combination of two genetic elements of the invention, the regulatory polynucleotide (PA) comprises or consists of a sequence of nucleotides ranging from nucleotide 1 to nucleotide 5906 of the sequence SEQ ID No. 1.

A functional regulatory polynucleotide (PA) that is included in a combination of two genetic elements according to the invention may also consist of a known promoter to direct the expression of the nucleic acid sequence encoding the ACS protein in a constitutive or tissue way specific.

A functional regulatory polynucleotide (PA) included in a combination of two genetic elements according to the invention can therefore be chosen from any one of the constitutive or tissue-specific promoters described above for certain embodiments of the regulatory polynucleotide (PG).

A functional regulatory polynucleotide (PA) according to the invention may also consist of an inducible promoter. Therefore, the invention aims at a combination of two genetic elements as defined above, in which the regulatory polynucleotide (PA) is sensitive to the action of an inducing signal and, preferably, in which the regulatory polynucleotide (PA ) is an inducible transcriptional or translational activator polynucleotide, which can be chosen from any one of the inducible activator polynucleotides described in certain embodiments of the regulatory polynucleotide (PG).

When this nucleic acid construct is used in a cell host, the expression of the polynucleotide encoding the ACS protein according to the invention can be induced by contacting the transformed cell host with the activating inducing signal to which the activating regulatory polynucleotide is direct or indirectly sensitive.

When the absence of expression of the polynucleotide encoding an ACS polypeptide is sought in this transformed cell host, it is then sufficient to eliminate or suppress the presence of the activating inducing signal to which the regular transcriptional or translational polynucleotide is sensitive.

The person skilled in the art will thus be able to draw on their general technical knowledge in the field of regulatory polynucleotides, in particular those active in plants, to define the constructions that conform to the definition of the previous embodiment and, in particular, those described for the regulatory polynucleotide (PG).

- Nucleic acids encoding the ACS protein

Preferably, the nucleic acid encoding the ACS protein comprises, from the 5 'end to the 3' end, at least:

(i) a sequence that has at least 95% identity with the polynucleotide ranging from nucleotide 9507 to nucleotide 6086 of the sequence SEQ ID No. 1,

(ii) A sequence that has at least 95% identity with the polynucleotide ranging from nucleotide 6181 to nucleotide 6467 of the sequence SEQ ID No. 1, and

(iii) a sequence that has at least 95% identity with the polynucleotide ranging from nucleotide 7046 to nucleotide 7915 of sequence SEQ ID N ° 1.

Genetic control element A / a, in the form of the recessive allele (a) of the combination according to the invention

In general, the control genetic element (A / a) in the form of a recessive allele (a), when present in a plant that does not have the dominant allele (A) in its genome, allows obtaining a level of ACS protein as elevated as that obtained when allele (A) is present.

Therefore, allele (a) can be defined as any alteration of the genotype corresponding to allele (A), which does not allow obtaining a level of active ACS.

The recessive allele (a) is distinguished from the dominant allele (A) in:

(i) a nucleic acid (NA) not present in the plant, or

(ii) a non-functional regulatory polynucleotide (Pa) in a dicotyledonous plant, or

(iii) a nucleic acid encoding a non-active ACS protein, or

(iv) a non-functional regulatory polynucleotide (Pa) in a dicotyledonous plant and a nucleic acid encoding a non-active ACS protein.

- Non-functional regulatory polynucleotide ( Pa)

A regulatory polynucleotide (Pa), or non-functional promoter according to the invention, is a nucleic acid that:

(i) does not allow the expression of the ACS protein of sequence SEQ ID N ° 3 in a host cell,

(ii) allows the expression of this protein at a low level compared to the level observed with the regulatory polynucleotide (PA), or

(iii) allows the expression of the ACS protein throughout the life of the plant, for a shorter period, compared to that observed with the regulatory polynucleotide (PA).

In certain embodiments of the non-functional regulatory polynucleotide (Pa), said polynucleotide (Pa) is methylated. For example, the regulatory polynucleotide (Pa) may consist of a regulatory polynucleotide (PA) that is present as a methylated nucleic acid.

By "methylated nucleic acid" or "methylated regulatory polynucleotide" is meant according to the invention, the corresponding nucleic acid whose ratio (number of methylated bases) / (number of unmethylated bases) is at least 5 / 1- Therefore, according to In the invention, a methylated nucleic acid encompasses a nucleic acid having a ratio (number of methylated bases) / (number of methylated bases n) of at least 6/1, 7/1, 8/1, 9/1, 10 / 1, 11/1, 12/1, 13/1, 14/1, 15/1, 16/1, 17/1, 18/1 and 20/1.

Determination of the ratio (number of methylated bases) / (number of unmethylated bases) can be done easily by one of ordinary skill in the art by any known technique. The person skilled in the art can particularly use the bisulfite sequencing method, as described in the examples of the present invention. To compare the expression level of various promoters, a simple technique, known to one of ordinary skill in the art, is to place a selection marker gene under the control of the promoters to be tested. A selection marker gene can be, for example, the BASTA herbicide resistance gene, well known to one of skill in the art.

Another technique may consist of measuring the level of ACS protein obtained when the sequence encoding this protein is under the control of different promoters, using antibodies directed to the encounter of this protein, and the procedures described in the part "polypeptides according to the invention".

As an example, a non-functional regulatory polynucleotide (Pa) comprises a nucleotide sequence ranging from nucleotide 1 to nucleotide 3650 of the sequence SEQ ID No. 2.

Also as an example, a non-functional regulatory polynucleotide (Pa) comprises certain nucleotide sequences that comprise one or more base substitutions, deletions or additions, with respect to the nucleotide sequence ranging from nucleotide 1 to nucleotide 3650 of the sequence SEQ ID No. 1.

Therefore, in the combination of two genetic elements for the control of the development of the floral type object of the invention, a non-functional regulatory polynucleotide (Pa) can comprise a nucleotide sequence ranging from nucleotide 1 to nucleotide 3650 of the sequence SEQ ID N ° 2 altered by one of the methods of the person skilled in the art.

The invention also has as its object a combination of two genetic elements for the control of the development of the floral type as defined in the present invention, and in which the allele (a) is a nucleic acid comprising the sequence SEQ ID N ° 2 This nucleic acid of sequence s Eq ID N ° 2 comprises a regulatory polynucleotide (Pa) and a nucleic acid that encodes the ACS protein of sequence SEQ ID N ° 3.

A non-functional polynucleotide (Pa) can also be any polynucleotide derived from the polynucleotide (PA) as defined above, the nucleotide sequence of which comprises an insertion, a substitution or a deletion of one or more nucleotides, with respect to the nucleotide sequence of the regulatory polynucleotide.

Therefore, in certain embodiments of the combination according to the invention, a polynucleotide (Pa) consists of a nucleic acid comprising a sequence of nucleotides that carries at least one alteration chosen from a mutation, an insertion or a deletion, with respect to to the nucleic acid that goes from nucleotide 1 to nucleotide 5907 of the sequence SEQ ID N ° 1, said altered nucleic acid leading to a reduced expression of the ACS protein, when it controls the expression of said protein, with respect to the expression of the ACS protein controlled by the nucleic acid ranging from nucleotide 1 to nucleotide 5907 of the sequence SEQ ID N ° 1. The invention also has for its object a combination of two genetic elements for the control of the development of the floral type as defined above, where the regulatory polynucleotide (Pa) is sensitive to the action of an inducing signal and, preferably, where the regulatory polynucleotide (Pa) is u n inducible transcription or translation repressor polynucleotide.

According to this first particular embodiment, the expression of the sequence encoding an ACS protein is constitutive in the chosen cell host, in the absence of the repressor inducing signal to which the repressor regulatory polynucleotide is directly or indirectly sensitive.

The contact of the cell host with the repressor inducing signal has the effect, thanks to a direct or indirect action on the repressor regulatory polynucleotide, to inhibit and / or block the expression of the polynucleotide encoding the ACS protein.

In order to carry out the DNA constructions according to the invention comprising a repressor regulatory polynucleotide, the person skilled in the art will be able to draw on his general technical knowledge in the field of gene expression in plants.

A procedure for obtaining a transformed plant, using this type of regulatory polynucleotide, is described in the part entitled "Procedures for obtaining a transformed plant according to the invention".

- Non-functional nucleic acid ( Na) for the expression of an active ACS protein

A nucleic acid (Na) encompasses nucleic acids that comprise at least a part of a sequence that encodes an active ACS protein but does not allow, when placed under the control of a functional regulatory polynucleotide in dicot plant cells, the production in such plants an active CmWIP1 protein.

A nucleic acid (Na) essentially encompasses nucleic acids (NA) in which one or more mutations are present in at least one intron or one exon, each mutation being chosen from (i) the substitution of one nucleotide or more than one nucleotide (ii) the deletion of a nucleotide or of at least two consecutive nucleotides and (ii) the deletion of a nucleotide or of at least two consecutive nucleotides, with respect to the nucleic acid (NA) of reference. A nucleic acid (Na) particularly encompasses nucleic acids that encode a non-active ACS protein.

By nucleic acid encoding a non-active ACS protein is meant, in the context of the present invention, a nucleic acid encoding a protein that differs from the ACS protein of sequence SEQ ID No. 3 by substitution, deletion or insertion of one or more amino acids and that does not possess the biological activity of the ACS protein of sequence SEQ ID No. 3. An illustrative example of this nucleic acid is presented in Figure 8.

In particular, this non-active ACS protein does not allow the transformation of S-adenosyl-methionine into ACC (1-aminocyclopropane-1-carboxylate).

Nucleic acids according to the invention

As previously indicated, two allelic variants (G) and (g) of the second control genetic element (G / g) included in a combination of two genetic elements for the control of flower development of the invention were characterized.

The inventors have identified the nucleic acid of sequence SEQ ID N ° 10 as a nucleic acid corresponding to the dominant allelic variant (G) and its methylated form in planta, as corresponding to the recessive allelic variant (g) of the second control genetic element in the form of a gene (G / g).

In the combination of two genetic elements for the control of flower development of the invention, at least the second of the two control genetic elements was artificially constructed in a plant.

As stated previously, this introduction causes a change of sex of the flower of the plant, which is one of the objectives sought according to the invention.

Consequently, the nucleic acid of sequence SEQ ID No. 10 forms part of the objectives of the invention.

The present invention therefore has as its object a nucleic acid that comprises a polynucleotide that has at least 95% nucleotide identity with the nucleotide sequence SEQ ID No. 10, or with a fragment of the sequence SEQ ID No. 10, provided that this nucleic acid possesses the functional characteristics of the (G) allele as defined above.

A nucleic acid with a sequence complementary to the nucleic acid as defined above is also part of the invention.

Another object of the invention is a nucleic acid consisting of a polynucleotide that has at least 95% nucleotide identity with the sequence SEQ ID No. 10, or with a fragment of the sequence SEQ ID No. 10, or an acid nucleic acid of complementary sequence, provided that this nucleic acid possesses the functional characteristics of the (G) allele as defined above.

The invention also relates to a nucleic acid comprising at least 12, preferably at least 15 and very preferably at least 20 consecutive nucleotides of the nucleic acid of sequence SEQ ID No. 10, it being understood that this nucleic acid encompasses in its definition the "fragments" of a nucleic acid according to the invention as defined in the present description.

The invention also relates to nucleic acid comprising or consisting of the sequence SEQ ID No. 10. The invention also relates to a nucleic acid comprising at least 12, preferably at least 15 and most preferably at least 20 nucleotides consecutive numbers of the nucleic acid of sequence SEQ ID No. 10, it being understood that this nucleic acid encompasses in its definition the "fragments" of a nucleic acid according to the invention as defined in the present description.

The allele (G) defined by the sequence SEQ ID N ° 10 comprises, at the 5 'end towards the 3' end, respectively:

a) a non-coding sequence that carries regulatory elements of transcription and / or translation of this gene, located upstream of the first exon, from nucleotide at position 1 to nucleotide at position 2999 of the sequence SEQ ID N ° 10 ;

b) a so-called "coding" region comprising the two exons and the intron of the gene (G / g), this coding region being located from the nucleotide at position 3000 to the nucleotide at position 5901 of the sequence SEQ ID No. 10; and

c) a non-coding region located below the coding region, which goes from nucleotide at position 5902 to nucleotide at position 7621 of the sequence SEQ ID No. 10.

The structural characteristics of the two exons and of the intron of the G / g gene are detailed in Table 2 below. The structural characteristics of the two exons and of the intron of the alleles (G) and (g) of the gene (G / g) are very similar, so that the exons of the alleles (G) and (g) can, in certain embodiments, encode the same protein of the sequence SEQ ID No. 12. As indicated above, the main difference between the nucleotide sequences corresponding to the alleles (G) and (g) can be found:

(i) either in the upward regulatory sequences corresponding to these two alleles, which are unmethylated in planta for allele (G) and which are methylated in planta for allele (g),

(ii) either in the sequence of the exons, since a single nucleotide substitution that causes the substitution of an amino acid in the sequence of the CmWIP1 protein is sufficient to obtain the allele (g)

Table 2

The invention also relates to a nucleic acid comprising at least 12 consecutive nucleotides of an exonic polynuclotide of the G / g gene, such as polynucleotides 1 and 2 described in Table 2 below, which are included in the nucleic acid of sequence SEQ ID No. 10.

This nucleic acid encodes at least a part of the CmWIP1 protein and can be inserted into a recombinant vector intended for the expression of the corresponding translation product in a host cell or in a plant transformed with this recombinant vector, in order to obtain a plant genotype (G).

This nucleic acid can also be used for the synthesis of nucleotide probes and primers intended for the detection or amplification of nucleotide sequences comprised in the gene (G / g) in a sample.

Where appropriate, the sequences described above may carry one or more mutations, preferably one or more mutations of a nature that induces the synthesis of a non-active CmWIP1 protein and that modifies the sexual type of a plant that carries this gene (G / g ) mutated. These sequences respond to the definition of nucleic acids that encode a non-active CmWIP1 protein, generally defined previously.

Table 3

The invention also relates to a nucleic acid comprising at least 12 consecutive nucleotides of an intronic polynucleotide of the gene (G / g), described in Table 3 above, which are included in the nucleic acid of sequence SEQ ID No. 10.

This nucleic acid can be used as an oligonucleotide probe or primer to detect the presence of at least one copy of the gene (G / g) in a sample or even to amplify a specific target sequence in the gene (A / a).

This nucleic acid can also be used to amplify a specific target sequence in the gene (G / g) or to inhibit it by a sense or co-deletion approach, or by using double-stranded RNA (Wassenegger et al. 1996; Kooter et al. 1999) for interference. This nucleic acid can also be used to search for functional allelic variants of the gene (G / g), which could be used in a method of selecting plants that have a specific sexual type.

Other nucleic acids according to the invention, encoding the CmWIP1 protein

The invention also has for its object a nucleic acid comprising a polynucleotide that has at least 95% nucleotide identity with the nucleotide sequence starting from the nucleotide at position 3000 and ending at the nucleotide at position 5901 of the sequence SEQ ID No. 10, as well as a nucleic acid of complementary sequence.

The invention also relates to a nucleic acid that has at least 95% identity in nucleotides with the nucleotide sequence starting from nucleotide at position 3000 and ending at nucleotide at position 5901 of the sequence SEQ ID N ° 10, as well as a nucleic acid of complementary sequence.

Another subject of the invention is a nucleic acid comprising the nucleotide sequence starting from nucleotide at position 3000 and ending at nucleotide at position 5901 of the sequence SEQ iD No. 10 or a nucleic acid of complementary sequence.

The invention also relates to a nucleic acid consisting of the nucleotide sequence starting from nucleotide at position 3000 and ending at nucleotide at position 5901 of the sequence SEQ ID No. 10 or a nucleic acid of complementary sequence.

Another object of the invention consists of a nucleic acid that comprises, at least:

(i) a sequence that has at least 95 identity with the polynucleotide ranging from nucleotide 3000 to nucleotide 3617 of the sequence SEQ ID No. 10,

(ii) a sequence that has at least 95% identity with the polynucleotide ranging from nucleotide 5458 to nucleotide 5901 of the sequence SEQ ID No. 10.

Another subject of the invention is a nucleic acid that comprises, from the 5 'end to the 3' end: (i) a sequence ranging from nucleotide 3000 to nucleotide 3617 of SEQ ID No. 10, and

(ii) a sequence ranging from nucleotide 5458 to nucleotide 5901 of SEQ ID No. 10.

A nucleic acid encoding the CmWIP1 protein may further comprise leader and terminator sequences, common to one of ordinary skill in the art.

Transcription and translation products of the gene (G / g) and polypeptides according to the invention

The expression of the genomic nucleic acid encoding the CmWIP1 protein leads to the synthesis of a messenger RNA whose cDNA is the nucleic acid of sequence SEQ ID No. 11, which is also one of the objects of the present invention.

Therefore, the invention has for its object the polypeptide that comprises the nucleic acid sequence SEQ ID No. 12, also called "CmWIP1 protein" in the present invention, as well as a polypeptide that has at least 95% amino acid identity with the sequence SEQ ID No. 12, a fragment or variant thereof.

An illustration according to the invention of a CmWIP1 protein that possesses at least 95% amino acid identity with the sequence SEQ ID No. 12 consists of the protein of sequence SEQ ID No. 16, which differs from the protein of sequence SEQ ID No. 12 by the deletion of a cerine residue.

A fragment of a CmWIP1 protein according to the invention comprises at least 10, 50, 100, 200, 300, 320, 330, 340, 345 or 353 consecutive amino acids of a polypeptide of sequence SEQ ID N ° 12.

The invention also relates to a polypeptide comprising a nucleic acid sequence that has at least 95% amino acid identity with the sequence of a CmWIP1 protein of SEQ ID No. 12 sequence. Advantageously, it also forms part of the invention a polypeptide that has at least 96% 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7 %, 99.8% or 99.9% identity of amino acids with the sequence of a polypeptide of sequence SEQ ID No. 12, or a polypeptide fragment of the latter.

In general, the polypeptides according to the present invention are presented in isolated or purified form.

A polypeptide according to the invention can be obtained by genetic recombination according to techniques well known to one skilled in the art, for example, the techniques described by AUSUBEL et al. (1989).

A polypeptide according to the invention can also be prepared by classical techniques of chemical synthesis, indifferently in homogeneous solution or in solid phase.

By way of illustration, a polypeptide according to the invention can be prepared by the homogeneous solution technique described by HOUBEN WEIL (1974) or even by the solid phase synthesis technique described by MERRIFIELD (1965a; 1965b).

Preferably, variant polypeptides of a polypeptide according to the invention retain their ability to be recognized by antibodies directed against polypeptides of sequences SEQ ID N ° 12.

A polypeptide encoded by the gene (G / g) according to the invention, such as a polypeptide of amino acid sequence SEQ ID No. 12, or even a variant or a peptide fragment of the latter is useful in particular for the preparation of antibodies intended for the detection of the presence and / or the expression of a polypeptide of sequences SEQ ID N ° 12 or of a peptide fragment of the latter in a sample.

In addition to the detection of the presence of a polypeptide encoded by the gene (G / g) or even a peptide fragment of this polypeptide in a sample, antibodies directed against these polypeptides are used to quantify the synthesis of a polypeptide of SEQ sequences ID N ° 12, for example, in cells of a plant and thus determine the sex of the plant, without therefore being necessary to cultivate it.

By "antibody" in the context of the present invention, it is meant in particular polyclonal or monoclonal antibodies or fragments (for example, the F (ab) ' ² , F (ab) fragments) or even any polypeptide comprising a domain of the antibody initial that recognizes the target polypeptide or polypeptide fragment according to the invention.

Monoclonal antibodies can be prepared from hybridomas according to the technique described by KOHLER and MILSTEIN (1975).

The present invention also relates to antibodies directed against a polypeptide as described above or a fragment or variant of the latter, such as the product in the trioma technique or even the hybridoma technique described by KOZBOR et al. (1983).

The invention also deals with Fv single chain antibody fragments (ScFv) as described in US Patent No. 4,946,778 or even by MARTINEAU et al. (1998).

The antibodies according to the invention also comprise antibody fragments obtained by means of phage libraries as described by RIDDER et al. (1995) or even humanized antibodies as described by REINMANN et al. (1997) and ^l E ^g ER et al. (1997). The antibody preparations according to the invention are useful in immunological detection assays aimed at identifying the presence and / or the amount of a polypeptide of sequences SEQ ID N ° 3, or of a peptide fragment thereof, present in a sample. .

An antibody according to the invention may comprise isotopies, an isotopic or non-isotopic detectable marker, for example fluorescent, or even be coupled to a molecule such as biotin, according to techniques well known to a person skilled in the art.

Therefore, the invention also has as its object a method for detecting the presence of a polypeptide according to the invention in a sample, said method comprising the steps of:

a) contacting the sample to be tested with an antibody as described above; b) detecting the antigen / antibody complex formed.

The invention also relates to a diagnostic test kit or kit for detecting the presence of a polypeptide according to the invention in a sample, said kit comprising:

a) an antibody as defined above;

b) where appropriate, one of several reagents necessary for the detection of the antigen / antibody complex formed.

Another object of the invention consists in the use of a nucleic acid or an allelic variant of a nucleic acid as defined above in plant selection programs to obtain plants whose floral type has been modified.

Nucleic acids comprising a functional regulatory polynucleotide (PG)

A functional promoter or regulatory polynucleotide (PG) according to the invention consists of a nucleic acid that allows the expression of the CmWIPI protein of sequence SEQ ID N ° 12 in dicotyledonous plants.

This functional regulatory polynucleotide (PG) thus makes it possible, when artificially introduced into a plant, to modify the sex of the flowers of this plant and, in particular, makes it possible to obtain male and female plants or males and hermaphrodites, capable of self-pollination.

Therefore, the invention also has as its object a nucleic acid that comprises a polynucleotide that has at least 95% nucleotide identity with the nucleotide sequence starting from the nucleotide at position 1 and ending at the nucleotide at position 2999 of the SEQ sequence ID No. 10, as well as a nucleic acid of complementary sequence.

The invention also relates to a nucleic acid that has at least 95% nucleotide identity with the nucleotide sequence starting from the nucleotide at position 1 and ending at the nucleotide at position 2999 of the sequence SEQ ID No. 10, as well as a nucleic acid of complementary sequence.

Another subject of the invention is a nucleic acid comprising the nucleotide sequence starting from the nucleotide at position 1 and ending at the nucleotide at position 2999 of the sequence SEQ ID No. 10 or a nucleic acid of complementary sequence.

The invention also concerns a nucleic acid consisting of the nucleotide sequence starting from the nucleotide at position 1 and ending at the nucleotide at position 2999 of the sequence SEQ ID No. 10 or of a nucleic acid of complementary sequence.

The regulatory polynucleotide having the nucleotide at position 1 to nucleotide at position 2999 of the sequence SEQ ID No. 10 is also referred to as the nucleic acid of sequence SEQ ID No. 13 in the present description. The invention also relates to a nucleic acid comprising at least 12 consecutive nucleotides of a regulatory polynucleotide, as defined above.

This nucleic acid can be used as an oligonucleotide probe or primer to detect the presence of at least one copy of the allele (G) or the gene (G / g) in a sample, to amplify a specific target sequence in the gene (G / g ). This nucleic acid can also be used to search for functional allelic variants of the gene (G / g) or it can be used in a method of selecting plants that have a specific sexual type.

The detection procedures using nucleic acids as described above are described in the part entitled "Selection procedures according to the invention".

This nucleic acid can be used to inhibit a specific target sequence in the gene (G / g) by an antisense or co-suppression approach or by using double-stranded RNA (Wassenegger et al.

nineteen ninety six; Kooter et al. 1999) for interference.

Nucleic acids comprising a non-functional Pg regulatory polypeptide

A non-functional promoter or regulatory polypeptide (Pa) according to the invention is a nucleic acid that:

(i) does not allow the expression of the CmWIP1 protein of sequence SEQ ID N ° 12 in a host cell, or (ii) allows the expression of this protein at a very low level compared to the level observed with the regulatory polynucleotide (PG ), or

(iii) it allows the expression of the CmWIP1 protein during the life of the plant, for a shorter period, compared to that observed with the regulatory polynucleotide (Pg).

This non-functional regulatory polynucleotide (Pg) allows, therefore, when it is artificially introduced into a plant, for example, in substitution of a polynucleotide (G), to modify the sex of the flowers of this plant and, in particular, to obtain plants hermaphrodites, capable of self-pollination or of female plants.

This nucleic acid can be used as an oligonucleotide probe or primer to detect the presence of at least one allele pair (a) of the gene (G / g) in a sample or even to amplify a specific target sequence in the gene (G / g ).

The invention finally relates to nucleic acids that comprise the association of one or more nucleic acids as defined above, for example, a nucleic acid that encodes a functional CmWIP1 protein under the control of a promoter of the (PG) or (Pg ).

General definitions

According to the invention, any common molecular biology, microbiology and recombinant DNA technique known to a person skilled in the art can be used. These techniques are described, for example, by SAMBROOK et al. (1989), GLOVER (1985), GAIT (1984), HAMES et HIGGINS (1984), BERBAL (1984) and AUSUBEL et al. (1994). Preferably, any nucleic acid and any polypeptide according to the invention is presented in isolated or purified form.

The term "isolated" in the context of the present invention indicates a biological material that has been removed from its original environment (the environment in which it is naturally located). For example, a polynucleotide naturally present in a plant is not isolated. The same polynucleotide separated from adjacent nucleic acids into which it is naturally inserted into the plant genome if isolated. This polynucleotide can be included in a vector and / or this nucleotide can be included in a composition and yet remain in an isolated state because the vector or composition does not constitute its natural environment.

The term "purified" does not specify that the material be present in an absolute purity form, which excludes the presence of other compounds. It is mostly a relative definition.

A polynucleotide or polypeptide is in a purified state after purification of the starting material or natural material of at least one order of magnitude, preferably two or three, and preferably four or five orders of magnitude.

For the purposes of the present description, the term "nucleotide sequence" can be used to indifferently denote a nucleotide or a nucleic acid. The term "nucleotide sequence" encompasses the genetic material itself and is therefore not restricted to information regarding its sequence.

The terms "nucleic acid", "polynucleotide", "oligonucleotide" or even "nucleotide sequence" encompass the sequences of RNA, DNA, cDNA or even hybrid RNA / DNA sequences of more than one nucleotide, indifferently in the form of single chain or duplex.

The term "nucleotide" indicates at the same time natural nucleotides (A, T, G, C), as well as modified nucleotides comprising at least one modification such as (i) an analog of a purine, (ii) an analog of a pyrimidine or (iii) a sugar analog, these modified nucleotides being described, for example, in PCT application No. WO95 / 04064.

For the purposes of the present invention, a first nucleotide is considered to be "complementary" to a second polynucleotide when each base of the first nucleotide is paired to the complementary base of the second polynucleotide whose orientation is reversed. The complementary bases are A and T (or A and U) and C and G.

According to the invention, a first nucleic acid that has at least 95% identity with a second reference nucleic acid will possess at least 95%, preferably at least 96%, 97%, 98%, 98.5%, 99 %, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% nucleotide identity with this second reference polynucleotide, the percent identity between two sequences being determined as described above.

The "percent identity" between two nucleic acid sequences, in the context of the present invention, is determined by comparing the two optimally aligned sequences, through a comparison window. The part of the nucleotide sequence in the comparison window can therefore comprise additions or deletions (for example, "gaps") with respect to the reference sequence (which does not comprise these additions or these deletions) so as to obtain a optimal alignment between the two sequences.

Percent identity is calculated by determining the number of positions in which an identical nucleic base is observed for the two sequences compared, then dividing the number of positions at which there is identity between the two nucleic bases by the total number of positions in the comparison window, then multiplying the result by 100 in order to obtain the percentage of nucleotide identity of the two sequences between them.

Optimal alignment of the sequences for comparison can be performed informally by means of known algorithms.

Especially preferably, the percentage of sequence identity is determined by means of the CLUSTAL W application (version 1.82), the parameters being set as follows: (1) CPU ^m O ^d E = ClustalW mp; (2) ALIGNMENT = "full"; (3) OUTPUT FORMAT = "aln w / numbers"; (4) OUTPUT ORDER = «aligned»; (5) COLOR ALIGNMENT = "no"; (6) KTUP (word size) = "default"; (7) WINDOW LENGTH = "default"; (8) SCORE TYPE = "percent"; (9) TOPDIAG = "default"; (10) PAIRGAP = "default"; (11) PHYLOGENETIC TREE / TREE TYPE = "None"; (12) MATRIX = "default"; (13) GAP OPEN = "default"; (14) END GAPS = "default"; (15) GAP EXTENSION = "default"; (16) GAP DISTANCES = "default"; (17) TREE TYPE = "cladogram" and (18) TREE GRAP DISTANCES = "hide".

A nucleic acid having at least 95% nucleotide identity with a nucleic acid according to the invention encompasses the "variants" of a nucleic acid according to the invention.

By "variant" of a nucleic acid according to the invention is meant a nucleic acid that differs from the reference nucleic acid by one or more substitutions, additions or deletions of a nucleotide, with respect to the reference nucleic acid. A variant of a nucleic acid according to the invention can be of natural origin, such as a naturally occurring allelic variant. This nucleic acid variant can also be a non-natural nucleic acid obtained, for example, by mutagenesis techniques.

In general, the differences between the reference nucleic acid and the nucleic acid "variant" are small, such that the reference nucleic acid and the nucleic acid variant have very similar nucleotide sequences and, in many regions, identical. Nucleotide modifications present in a nucleic acid variant can be silent, meaning that they do not affect the amino acid sequence that may be encoded by this nucleic acid variant.

Nucleotide modifications in the nucleic acid variant can also result from substitutions, additions or deletions of one or more amino acids in the polypeptide sequence that can be encoded by this nucleic acid variant.

Especially preferably, a nucleic acid variant according to the invention comprises an open reading frame, encoded for a polypeptide that retains the same function or the same biological activity as the polypeptide encoded by the reference nucleic acid.

Especially preferably, a nucleic acid variant according to the invention comprising an open reading frame, codes for a polypeptide that retains the ability to be recognized by antibodies directed against the polypeptide encoded by the reference nucleic acid.

The nucleic acids of the genes orthologous to the CmWIP1 protein included in the plant genome and which have a nucleotide identity of at least 95% with a nucleic acid that are part of the "variants" of a nucleic acid encoding the ACS protein. encodes the CmWIP1 protein. By "Fragment" of a nucleic acid according to the invention is meant a sequence of nucleotides of reduced length with respect to the reference nucleic acid, the nucleic acid fragment having a nucleotide sequence identical to the nucleotide sequence of the reference nucleic acid on the common part.

These fragments of a nucleic acid according to the invention have at least 12, 15, 18, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 1000, 2000 or 3000 consecutive nucleotides of the reference nucleic acid, it being understood that the maximum nucleotide length of a fragment of a nucleic acid according to the invention is limited by the maximum nucleotide length of the reference nucleic acid.

Probes and primers

The nucleic acids according to the invention and, in particular, the nucleotide sequences SEQ ID N ° 10 and SEQ ID N ° 11, their fragments of at least 12 nucleotides, the regulatory polynucleotides (PG) and (Pg), as well as the acids nucleic acids of complementary sequence are useful for detecting the presence of at least one copy of a nucleotide sequence of the gene (G / g) or even of a fragment or of an allelic variant of the latter in a sample.

In particular, the above probes and primers derived from the sequence SEQ ID No. 10 and, in particular, derivatives of the regulatory polynucleotide (PG) can be used to detect the presence of the (G) allele in a dicotyledonous plant.

Also forming part of the invention are hybridizing nucleotide probes and primers, under high stringency hybridization conditions, with a nucleic acid chosen from the sequences SEQ ID N ° 10 and SEQ ID N ° 11 or with a regulatory polynucleotide (PG) or ( Pg).

Therefore, the invention also relates to a nucleic acid used as a probe or primer, which specifically hybridizes with a nucleic acid as defined above.

Subsequent hybridization conditions are used for hybridization of a nucleic acid, probe or primer, 20 bases in length.

The level and specificity of hybridization depend on different parameters, such as:

a) the purity of the nucleic acid preparation to which the probe or primer is to hybridize; b) the base composition of the probe or primer, the G-C base pairs that have greater thermal stability than the A-T or A-U base pairs;

c) the length of the homologous base sequence between the probe or primer and the nucleic acid;

d) the ionic strength: the degree of hybridization increases with the growth of the ionic strength and the duration of the incubation time;

e) incubation temperature;

f) the nucleic acid concentration to which the probe or primer should hybridize;

g) the presence of denaturing agents such as agents that promote the breaking of hydrogen bonds such as formamide or urea that increase the astringency of hybridization;

h) the incubation time, increasing the degree of hybridization with the duration of the incubation;

i) the presence of bulk exclusion agents, such as dextran sulfate dextran, which increase the degree of hybridization by increasing the effective concentrations of the probe or primer and nucleic acid to be hybridized, in the context of the preparation.

The parameters that define the stringency conditions depend on the temperature at which 50% of paired bonds separate (Tm).

For sequences comprising more than 360 bases, the Tm is defined by the relationship:

Tm = 81.5 0.41 (% G + C) 16.6 Log (cation concentration) - 0.63 (% formamide) - (600 / number of bases)

(SAMBROOK et al., (1989), pages 9.54-9.62).

For sequences shorter than 30 bases, the Tm is defined by the relationship: Tm = 4 (G + C) 2 (A + T). Under the appropriate stringency conditions, in which specific sequences do not hybridize, the hybridization temperature is about 5 to 30 ° C, preferably 5 to 10 ° C below the Tm. By "high stringency hybridization conditions" according to the invention is meant hybridization conditions such that they are placed at a hybridization temperature of 5 ° C below the Tm.

The hybridization conditions described above can be adapted as a function of the length and base composition of the nucleic acid whose hybridization is sought or the type of labeling chosen, according to techniques known to a person skilled in the art.

The suitable hybridization conditions can be adapted, for example, according to the instructions contained in the publication of HAMES and HIGGINS (1985) or even in the publication of AUSUBEL et al. (1989).

By way of illustration, the hybridization conditions used for nucleic acid 200 bases in length are as follows:

Prehybridization:

Same conditions as for hybridization

Duration: 1 night.

Hybridization:

5 x SSPE (0.9 M NaCl, 50 mM sodium phosphate, pH 7.7, 5 mM EDTA)

5 x Denhardt's (0.2% PVP, 0.2% Ficoll, 0.2% SAB)

10 pg / ml salmon sperm DNA

0.1% SDS

Duration: 1 night.

Washes:

2 x SSC, 0.1% SDS, 10 min, 65 ° C

I x SSC, 0.1% SDS, 10 min, 65 ° C

0.5 x SSC, 0.1% SDS, 10 min, 65 ° C

0.1 x SSC, 0.1% SDS, 10 min, 65 ° C.

The probes or nucleotide primers according to the invention comprise at least 12 consecutive nucleotides of a nucleic acid according to the invention, in particular a nucleic acid of sequences SEQ ID N ° 10 or SEQ ID N ° II or their complementary sequence, of a nucleic acid that has 95% nucleotide identity with a sequence chosen from the sequences SEQ ID N ° 10 or SEQ ID N ° 11 or their complementary sequence or even a hybridizing nucleic acid under high stringency hybridization conditions with a sequence chosen from the sequences SEQ ID N ° 10 or SEQ ID N ° 11 or its complementary sequence.

Preferably, the nucleotide probes or primers according to the invention will have a length of at least 12, 15, 18, 20, 25, 30, 35, 40, 45, 50, 60, 100, 150, 200, 300, 400, 500 , 1000, 2000 or 3000 consecutive nucleotides of a nucleic acid according to the invention.

Alternatively, a nucleotide probe or primer according to the invention will consist of and / or comprise fragments with a length of 12, 12, 15, 18, 20, 25, 30, 35, 40, 45, 50, 60, 100, 150, 200, 300, 400, 500, 1000, 2000 or 3000 consecutive nucleotides of a nucleic acid according to the invention.

A nucleotide primer or probe according to the invention can be prepared by any adapted method well known to one skilled in the art, including cloning and action of restriction enzymes or even by direct chemical synthesis according to techniques such as the phosphodiester method of NARANG et al. (1979) or BROWN et al. (1979), the diethylphosphoramidite method of BEAUCAGE et al. (1980) or even the solid support technique described in European Patent No. EP 0,707,592. Each of the nucleic acids according to the invention, including the oligonucleotide probes and primers described above, can be labeled if desired, incorporating a detectable molecule, that is to say a marker detectable by spectroscopic, photochemical, biochemical, immunochemical or even chemical means.

For example, these markers can consist of radioactive isotopes (32P, 3H, 35S) of fluorescent molecules (5-bromo deoxyuridine, fluorescein, acetylaminofluorene or even ligands such as biotin).

The labeling of the probes is preferably done by incorporating labeled molecules into polynucleotides by extension of primers or by addition to the 5 'or 3' ends.

Examples of non-radioactive labeling of nucleic acid fragments are described in particular in French patent No. FR 7810975 or even in the articles of URDEA et al. (1988) or SANCHEZ PESCADOR et al. (1988).

Advantageously, the probes according to the invention can have structural characteristics of a nature that allow amplification of the signal, such as the probes described by URDEA et al; (1991) or even in European Patent No. EP 0225807 (Chiron).

The oligonucleotide probes according to the invention can be used in particular in Southern type hybridizations in any nucleic acid that encodes the protein SEQ ID No. and, in particular, nucleic acids of sequences SEQ ID No. 10 or SEQ ID No. 11 or even in RNA hybridizations when looking for the expression of the corresponding transcript in a sample.

The probes according to the invention can also be used for the detection of PCR amplification products or even for the detection of mismatches.

The nucleotide probes or primers according to the invention can be immobilized on a solid support. These solid supports are well known to one skilled in the art and comprise well surfaces of microtiter plates, polystyrene beads, magnetic beads, nitrocellulose bands, or even microparticles such as latex particles.

Consequently, the invention still has as its object a nucleic acid usable as a nucleotide probe or arbor, characterized in that it comprises at least 12 consecutive nucleotides of a nucleic acid as defined above, in particular, a nucleic acid consequences of nucleotides SEQ ID N ° 10 and SEQ ID N ° eleven.

The invention also relates to a nucleic acid usable as a nucleotide probe or primer, characterized in that it consists of a polynucleotides of at least 12 consecutive nucleotides of a nucleic acid according to the invention, in particular a nucleic acid of sequences chosen from among the nucleotide sequences SEQ ID No. 10 and SEQ ID No. 11.

As described above, this nucleic acid can be further characterized in that it is labeled with a detectable molecule.

A nucleic acid usable as a nucleotide probe or primer for detection or amplification of a genomic, mRNA or cDNA sequence of the gene (G / g) can be further characterized in that it is chosen from the following sequences:

a) hybridizing nucleotide sequences, under high stringency hybridization conditions, with a nucleic acid of sequence SEQ ID N ° 10 or SEQ ID N ° 11;

b) sequences comprising at least 12 consecutive nucleotides of a nucleic acid of sequence SEQ ID N ° 10 or SEQ ID N ° 11.

Vectors, cells and plants according to the invention

In the combination of two genetic elements for the control of the development of the floral type of a dicotyledonous plant object of the invention, to allow that at least one of the control genetic elements is artificially introduced into the dicotyledonous plant, the nucleic acids and the polynucleotides Regulators defined above must be introduced into vectors and subsequently into cells.

Therefore, the invention also relates to transformed vectors, cells and plants, comprising regulatory polynucleotides (PG) and (Pg), nucleic acids encoding active or non-active CmWIP1 proteins, as well as nucleic acids corresponding to alleles (G) and (g) as defined above and the primers as defined therein.

Cartoon vector

A nucleic acid as defined above, hereinafter referred to as nucleic acid of interest, can be inserted into an appropriate vector.

By "vector" in the context of the present invention, it is meant a circular or linear DNA or RNA molecule that is indifferently in single-stranded or double-stranded form.

A recombinant vector according to the invention is preferably an expression vector or, more specifically, an insertion vector, a transformation vector or an integration vector.

It can in particular be a vector of bacterial or viral origin.

In all cases, the nucleic acid of interest is placed under the control of one or more sequences that contain regulatory signals of its expression in the plant in question, either regulatory signals, that is, the regulatory signals are all contained in the nucleic acid of interest, as is the case in the nucleic acid constructs described in the previous section, that is, one or more of them, or even all the regulatory signals are contained in the receptor vector in which it has been inserted the nucleic acid of interest.

A recombinant vector according to the invention advantageously comprises appropriate transcription start and stop sequences.

Furthermore, the recombinant vectors according to the invention may include one or more functional origins of replication in the host cells in which their expression is sought, as well as, where appropriate, sequences of selection marker nucleotides.

Recombinant vectors according to the invention may also include one or more expression regulation signals as defined above in the description.

Preferred bacterial vectors according to the invention are, for example, vectors pBR322 (ATCC No. 37 017) or even vectors such as pAA223-3 (Pharmacia, Uppsala, Sweden) and pGEM1 (Promega Biotech, Madison, USA).

Still other marketed vectors can be mentioned such as the vectors pQE70, pQE60, pQE9 (Quiagen), psiX174, pBluescript SA, pNH8A, pMH16A, pMH18A, pMH46A, pWLNEO, pSV2CAT, pOG44, pXTI and pSG (Stratagene).

It can also be Baculovirus- type vectors such as the vector pVL1392 / 1393 (Pharmingen) used to transfect cells of the Sf9 line (ATCC No. CRL 1711) derived from Spodoptera frugiperda.

Preferably, and for the main application of vectors of the invention consisting in obtaining a stable and preferably inducible expression of a sequence encoding a CmWIP1 protein in a plant, specially adapted vectors will be used for the expression of sequences of interest in plant cells, such as the following vectors:

- vector pBIN19 (BEVAN et al.), commercialized by the company CLONTECH (Palo Alto, Californie, USA);

- vector pBI 101 (JEFFERSON, 1987), marketed by the company CLONTECH;

- vector pBI121 (JEFFERSON, 1987), marketed by the company CLONTECH;

- vector pEGFP; Yang et al. (1996), marketed by the company CLONTECH;

- vector pCAMBIA 1302 (HAJDUKIIEWICZ et al., 1994)

- intermediate and superbinadiro vectors derived from the vectors pSB12 and pSB1 described by Japan Tobacco (EP 672752 by Ishida et al., 1996).

Cells

The most widespread methods for introducing nucleic acids into bacterial cells can be used in the context of this invention. It may be the function of receptor cells with bacterial protoplasts containing DNA, electroporation, projectile bombardment, infection by viral vectors, etc. Bacterial cells are often used to amplify the number of plasmids that contain the construct comprising the nucleotide sequence, object of the invention. The bacteria are cultured and the plasmids are then isolated according to methods well known to one skilled in the art (reference is made to the protocol manuals already cited), including commercially available plasmid purification test kits, for example, by EasyPrepl from Pharmacia Biotech or Qiagen's QIAexpress Expression System. The plasmids thus isolated and purified are then manipulated to produce other plasmids that will be used to transfect plant cells.

To allow the expression of a nucleic acid of interest according to the invention placed under the control of an appropriate regulatory sequence, the nucleic acids or the recombinant vectors defined in the present invention must be introduced into a host cell. The introduction of the polynucleotides according to the invention into a host cell can be carried out in vitro according to techniques well known to a person skilled in the art. The invention furthermore relates to a host cell transformed by a nucleic acid according to the invention or by a recombinant vector as defined above.

This transformed host cell is preferably of bacterial, fungal or plant origin.

Therefore, bacterial cells from different strains of Escherichia coli or even Agrobacterium tumefaciens can be used .

Advantageously, the transformed host cell is a plant cell or even a plant protoplast. Among the cells that can be transformed according to the method of the invention, one can cite as examples dicotyledonous plant cells, preferably belonging to the cucurbit family, the members of which are detailed below in the part entitled "Plants according to the invention".

Preferably, it is a cell or a protoplast of a plant belonging to the species Cucumis melo. The invention also has as its object the use of a nucleic acid of interest to manufacture a transformed plant whose sexual phenotype is modified.

The invention also relates to the use of a recombinant vector as defined in the present description to manufacture a transformed plant whose sexual phenotype is modified.

The invention also relates to the use of a cell host transformed by a nucleic acid of interest, to manufacture a transformed plant whose sexual phenotype is modified.

The invention also relates to a transformed plant comprising a plurality of host cells as defined above.

Plants transformed according to the invention.

The invention also relates to a transformed plant multicellular organism, characterized in that it comprises a transformed host cell or a plurality of host cells transformed by at least one of the nucleic acids as defined above or even by a recombinant vector comprising this nucleic acid .

The transformed plant may contain a plurality of copies of a nucleic acid encoding the CmWIP1 protein, in situations where an overexpression of the CmWIP1 protein is sought. An overexpression of the CmWIP1 protein is particularly sought when it is desired to obtain plants that produce male and female flowers or plants that produce male and hermaphroditic flowers.

A plant that overexpresses the CmWIP1 protein produces male and female flowers in the embodiments of the combination of two genetic elements of the invention in which the CmWIP1 protein is also overexpressed (allele A present in the homozygous or heterozygous and heterozygous state).

A plant that overexpresses the CmWIP1 protein produces male and hermaphroditic flowers in the embodiments of the combination of two genetic elements of the invention in which there is no expression of an active ACS protein.

Therefore, the invention also relates to a transformed plant as defined above whose flowers are male and female and a transformed plant as defined above whose flowers are male and hermaphroditic.

The transformed plant may contain a plurality of copies of a nucleic acid encoding the ACS protein, in situations where an overexpression of the ACS protein is sought. An overexpression of the ACS protein is particularly sought when it is desired to obtain plants that produce female flowers, not capable of self-pollination.

A plant that overexpresses the ACS protein produces male and female flowers in the embodiments of the combination of two genetic elements of the invention in which the CmWIP1 protein is also overexpressed (G allele present in the homozygous state).

A plant that overexpresses the ACS protein produces female flowers in the embodiments of the combination of two genetic elements of the invention in which there is no expression of an active CmWIP1 protein.

Therefore, the invention also relates to a transformed plant as defined above whose flowers are male and female and to a transformed plant as defined above whose flowers are exclusively female.

The plants transformed according to the invention all comprise at least one second genetic element of the combination according to the invention, chosen from among nucleic acids and regulatory polynucleotides defined above in a form artificially introduced into their genome.

The invention also relates to any part of a transformed plant as defined in the present description as the root, but also to the area parts such as the stem, the leaves, the flower and especially the grain or the fruit. Another subject of the invention is a plant seed or grain produced by a transformed plant as defined above.

Normally, this transformed seed or this transformed grain comprises one or more cells that comprise in their genome one or more copies of the first and second genetic control elements as defined above, artificially introduced into said dicotyledonous plant, which allow the synthesis of the CmWIP1 protein at a high or low level, where appropriate in a controlled and inducible manner.

According to a preferred embodiment of a transformed plant according to the invention, the aim is to express the CmWIP1 protein in a controlled manner, which implies that the transformed plant does not contain, as a functional copy of a polynucleotide encoding the CmWIP1 protein, only the copy (s) that have been artificially introduced into its cells and, preferably, into its genome, while the sequences of the gene (G / g) encoding CmWIP1, which are found naturally in the wild plant, carry at least one mutation that causes a detect in the expression of the gene (G / g).

The plants transformed according to the invention are dicotyledonous, preferably belonging to the cucurbit family and, in particular, to the genera chosen from:

Abobra, Acanthosicyos, Actinostemma, Alsomitra, Ampelosicyos, Anacaona, Apatzingania, Apodanthera, Bambekea, Benincasa, Biswarea, Bolbostemma, Brandegea, bryonia, Calycophysum, Cayaponia, Cephalopentandra, Ceratosanthes, Coralluscarpus, Ceratosanthes, Coaluuxiagnus, Corallosemaccinia, Corallosemaccinia, Corallosemaccinia, Ceratosanthes, Corallus Ctenolepis, Cucumella, Cucumeropsis, Cucumis, Cucurbita ,, Cucurbitella, Cyclanthera, Cyclantheropsis, Dactyliandra, Dendrosicyos, Dicoelospermum, Dieterlea, Diplocyclos, Doyerea, Ecballium, Echinocystis, Echinopeponioville, Guranio, Ggarhone, Guranioville, Guranio, Ggarnia, Gurania , Gymnopetalum, Gynostemma, Halosicyos, Hanburia, Helmontia, Hemsleya, Herpetospermum, Hodgsonia, Ibervillea, Indofevillea, Kedrostis, Lagenaria ,, Lemurosicyos, Luffa, Marah, Melancium, Melothria, Melothrianthus, Microsechuelle, Momicyrgia, Murargia, Murargia, Murargia, Murargia, Murargia Nothoalsomitra, Odosicyos, Oreosyce, Parasicyos, Penelopeia, Peponium, Peponopsis, Polyclathra, Pos adaea, Praecitrullus, Pseudocyclanthera, Pseudosicydium, Psiguria, Pteropepon, Pterosicyos, Raphidiocystis, Ruthalicia, Rytidostylis, Schizocarpum, Schizopepon, Sechiopsis, Sechium ,, Selysia, Seyrigia, Sicana, Siicyuraitia, Sicynaramania, Sicana, Siicyolmania, Tecfairia, Siicyuraitia, Sicyuraitia, Sicana, Sicydium , Thladiantha, Trichosanthes, Tricyclandra, Trochomeria, Trochomeriopsis, Tumamoca, Vaseyanthus, Wilbrandia, Xerosicyos, Zanonia, Zehneria, Zombitsia, or Zygosicyos.

Preferably, the transformed plants belong to the genus Cucumis and to the species Cucumis Melo.

Detection procedures according to the invention

The identification of the floral type development control system by the inventors has made it possible to carry out extremely simple procedures for detecting the sexual phenotype of plants, the main characteristics of which are detailed below.

The invention also relates to a method for detecting the presence of a (G) or (g) allele, said method comprising the steps of:

1) Contacting a probe or a plurality of nucleotide probes as defined above with the sample to be tested; and

2) detect the complex eventually formed between the probe (s) and the nucleic acid present in the sample. The above detection procedure can be used in conjunction with the procedure for detecting the presence of an (A) or (a) allele described in PCT application No. WO 2007/125264, which comprises the steps of: 1) putting into contacting a probe or a plurality of nucleotide probes as defined above with the sample to be tested; and

2) detect the complex eventually formed between the probe (s) and the nucleic acid present in the sample. These two detection procedures allow the selection of plants whose phenotypes and genotypes are summarized in Table 1.

The detection of the complex between a nucleic acid and a probe can be done by any technique known to a person skilled in the art and, in particular, using labeled probes or primers, as described in the "probes and primers according to the invention" part.

These procedures are particularly advantageous since they avoid having to cultivate a dicotyledonous plant to determine its sexual phenotype. It is thus possible to carry out the detection of the sexual phenotype of a very large sample of plants at a lower cost.

Selection procedures according to the invention.

The above detection procedures can be used in the selection procedures detailed below.

Another object of the invention is a procedure for selecting the floral type of a plant belonging to the genus Cucurbitaceae, characterized in that it comprises the steps of:

a) Determine the presence of alleles (G) and (g) in a plant of interest belonging to the Cucurbit family, for example, using nucleic acids as defined above, and

b) positively select the plant that has allele (G) or allele (g) in its genome.

The above selection procedure can be used in conjunction with the selection procedure of the floral type of a plant belonging to the genus Cucurbitaceae described in PCT application No. WO 2007/125264, characterized in that it comprises the steps of:

a) determine the presence of alleles (A) and (a) in a plant of interest belonging to the cucurbit family, for example, using nucleic acids as defined above or an antibody directed against the ACS protein and

b) positively select the plant that has allele (A) or allele (a) in its genome.

The determination of the presence of the alleles (A), (a), (G) and (g) can therefore be advantageously carried out by carrying out the above detection procedures.

The person skilled in the art can easily combine the above-defined selection procedures, referred to in Table 1, which provide the correspondence between genotype and phenotype, to obtain plants with only female or hermaphroditic phenotypes, for example.

Procedure for obtaining a transformed plant according to the invention

The plants transformed according to the invention can be obtained thanks to any one of the numerous processes that are currently part of the general knowledge of a person skilled in the art.

Procedures for obtaining transformed plants according to the invention are described below.

Procedure for obtaining transformed plants by "TILLING"

TILLING (for "Targeted Induced Local Lésions In Genomes") is an inverse genetics method that is based on the ability of an endonuclease to detect mismatches in a DNA double bond and perform a DNA disconnection at the level of the unpaired bases. This technique allows the detection of unique mutation points generated by the exposure of plants to a mutagenic chemical compound. The TILLING technique therefore allows the identification of a series of alleles of a given gene and is particularly well adapted for the use of high-throughput selection procedures that allow the selection, in target genes of interest, of mutations induced by chemical mutagenesis.

The TILLING method is well known to one of ordinary skill in the art, it is particularly described by Mc Callum et al. (2000, Plant Physiology, Vol. 123: 439-442).

For the needs of the present invention, the TILLING technique is particularly well adapted to obtaining and selecting plants into which the allele (g) of the second genetic control element (G / g) has been artificially introduced.

According to the invention, the TILLING technique can also be used successfully to obtain and select plants in which the allele (a) of the first genetic element (A / a) has been artificially introduced.

By definition, considering the above, the TILLING technique is adapted for obtaining and selecting plants in which the allele (g) of the second genetic control element (G / g) and the second genetic control element (G / g) have been artificially introduced at the same time. allele (a) of the first genetic element (A / a).

Obtaining dicotyledonous plants artificially mutated in the gene encoding the CmWIP1 protein can be carried out using the TILLING technique, for example, according to a method comprising the following steps: a) generating a collection of mutant dicotyledonous plants by chemical mutagenesis;

b) selecting from the collection of mutant plants generated in step a) the plants that have one or more than one mutation in the gene encoding the CmWIP1 protein,

c) selecting, among the mutant plants selected in step b), the plants that express the phenotype (g). In certain embodiments of the above procedure, step a) is carried out by chemical mutagenesis of seeds of dicotyledonous plants of interest, by exposing the seeds to a mutagenic agent, for example, ethyl-methanesulfonate, for example, using the method described by Koornbeef et al. (1982, Mutat Res, Vol.

93: 109-123).

Next, a collection of M1 plants is generated from seeds previously exposed to the agent. mutagenic. The M1 plants are then selfed in order to generate a collection of M2 plants, which is the collection of plants generated in step a) of the procedure.

Next, in step b), the DNA of each plant is extracted from the plant collection generated in step a) and an amplification of the nucleic acid of the target gene is carried out, in this case the gene that encodes the CmWIP1 protein, and the presence of mutation (s) in the sequence of the target gene is searched for, by comparison with the sequence of the non-mutated target gene. Next, the mutated plants are selected in the sequence of the gene encoding the target gene, in this case the gene encoding the CmWIP1 protein.

In certain embodiments of step b), the DNA is extracted from several M2 plants, for example, 20 M2 plants and mixed initially and the detection of mutations in the target gene sequence is carried out on the mixture ("pool") of DNA extract in order to reduce the number of mutation detection steps that must be carried out.

Next, a step of amplifying the target sequences by PCR is carried out, using appropriate nucleic primers and the generated amplicons are heated, then cooled in order to generate DNA heteroduplexes between the original DNA of a non-mutated plant on the nucleic acid. target and DNA from a mutated step on the target nucleic acid.

The DNA heteroduplexes are then incubated in the presence of an endonuclease that cleaves at the level of the mismatches. The cleaved heteroduplexes are then denatured and separated.

The separated DNA strands are then subjected to the stage of detecting the mutation (s) themselves, for example, by electrophoresis or by HPLC under denaturing conditions (DHPLc).

In certain embodiments of step b), the detection of mutation (s) in the target gene is carried out using the HPLC technique under denaturing conditions ("DHPLC"), as described, for example, by Mc Callum et al. .

(2000, Plant Physiol., Vol. 123: 439-442).

Next, in step c), among the plants mutated on the target gene, in this case the plants mutated in the gene encoding the CmWIP1 protein, the plants that express the phenotype associated with allele (g) of the genetic element are selected (G / g).

As already mentioned previously, the TILLING technique can be used, according to the present invention, to obtain transformed plants that comprise any one of the combinations of alleles (G / g) and (A / a) described herein. invention.

Consequently, in certain embodiments of the above procedure, plants (i) that have one or more than one mutation in the gene encoding the CmWIP1 protein and (ii) that possess a mutation or more than one mutation in the gene encoding the ACS protein.

In these particular embodiments, it is then selected in step c), among the plants mutated on the two target genes, that is, the plants mutated in the gene that encodes the CmWIP1 protein and in the gene that encodes the ACS protein, the plants that express at the same time the phenotype associated with allele (g) of the genetic element (G / g) and the phenotype associated with allele (a) of the genetic element (A / a).

Another subject of the present invention is dicotyledonous plants of a modified floral type in the genome in which at least one mutation of the gene encoding the CmWIP1 protein has been introduced.

The present invention also relates to dicotyledonous plants of a modified floral type that have been artificially mutated in the sequence of the gene that encodes the CmWIP1 protein, said plants expressing the phenotype associated with allele (g) of the genetic element (G / g).

The subject of the present invention is also dicotyledonous plants of a modified floral type in the genome in which (i) at least one mutation has been introduced in the gene encoding the CmWIP1 protein and (ii) at least one mutation in the gene encoding the ACS protein.

The present invention also relates to dicotyledonous plants of a modified floral type that have been artificially mutated (i) in the sequence of the gene encoding the CmWIP1 protein and (ii) in the sequence of the gene encoding the ACS protein, said plants expressing at the same time (i) the phenotype associated with the allele (g) of the genetic element (G / g) and the phenotype associated with the allele (a) of the genetic element (G / g).

Other procedures for obtaining transformed plants

The invention relates first of all to a process for obtaining a transformed plant intended to insert the (G) allele into a plant that does not comprise this allele.

The invention therefore has as its object a process for obtaining a transformed plant, belonging to the cucurbit family, bearing female flowers, characterized in that it comprises the following stages:

a) transformation of at least one plant cell of a plant of interest that does not carry the allele (G) in its genome, by a nucleotide sequence (NG); or a recombinant vector comprising this nucleic acid;

b) selection of the transformed cells obtained in step a) that have integrated into their genome, nucleic acid (NG);

c) regeneration of a transformed plant from the cells transformed in step b).

This type of procedure is particularly useful since it allows the (G) allele to be inserted into the genome of a plant, thus presenting a monoecious or andromonoic phenotype.

Another object of the invention is a process for transforming plants intended to suppress the allele (G) in a plant or to replace the allele (G) with an allele (g), so as to obtain a plant with a hermaphroditic phenotype or a plant female type.

The object of the invention is therefore a process for obtaining a transformed plant belonging to the cucurbit family bearing hemaphrodite or female flowers, characterized in that it comprises the following stages:

a) the substitution in a plant of the allele (G) by an allele (g),

Another subject of the present invention is dicotyledonous plants of a modified floral type in the genome in which at least one mutation of the gene encoding the CmWIP1 protein has been introduced.

The present invention also relates to dicotyledonous plants of a modified floral type that have been artificially mutated in the sequence of the gene that encodes the CmWIP1 protein, said plants expressing the phenotype associated with allele (g) of the genetic element (G / g).

Another subject of the present invention is dicotyledonous plants of a modified floral type in the genome in which (i) at least one mutation has been introduced in the gene encoding the CmWIP1 protein (ii) at least one mutation in the gene encoding the ACS protein.

The present invention also relates to dicotyledonous plants of a modified floral type that have been artificially mutated (i) in the sequence of the gene encoding the CmWIP1 protein and (ii) in the sequence of the gene encoding the ACS protein, said plants expressing same (i) the phenotype associated with allele (g) of the genetic element (G / g) and the phenotype associated with allele (a) of the genetic element (G / g).

Other procedures for obtaining transformed plants

The invention relates first of all to a process for obtaining a transformed plant aimed at inserting the (G) allele into a plant that does not comprise this allele.

Therefore, the object of the invention is a process for obtaining a transformed plant, belonging to the cucurbitaceae family, bearing female flowers, characterized in that it comprises the following steps:

a) transformation of at least one plant cell of a plant of interest that does not carry the allele (G) in its genome, by a nucleotide sequence (NG); or a recombinant vector comprising this nucleic acid;

b) selection of the transformed cells obtained in step a) that have integrated nucleic acid (NG) into their genome;

c) regeneration of a transformed plant from the transformed cells obtained in step b).

This type of procedure is particularly useful since it allows the (G) allele to be inserted into the genome of a plant, thus presenting a monoecious or andromonoic phenotype.

Another object of the invention is a process for transforming plants intended to suppress the allele (G) in a plant or to replace the allele (G) with an allele (g) so as to obtain a plant with a hermaphroditic phenotype or a plant of female type.

Therefore, the object of the invention is a process for obtaining a transformed plant, belonging to the cucurbit family, which bears hermaphrodite flowers or female flowers, characterized in that it comprises the following stages:

a) the substitution in a plant of the allele (G) by an allele (g),

b) selection of the transformed cells obtained in step (a) that have integrated the allele (g) into their genome; c) regeneration of a transformed plant from the transformed cells of step b).

Without this forming part of the invention, the process may still comprise a stage d) of growth of plants obtained in stage c) to obtain one that no longer carries the (G) allele.

In a first embodiment of the above procedure, step a) consists of transforming a plant that comprises the allele (G) in its genome by an "antisense" nucleic acid as defined above, and selecting the plants that already they do not understand the (G) allele.

An identical result can be obtained using homologous recombination phenomena aimed at replacing all or part of the nucleic acid (NG) by a nucleic acid with an altered structure, which does not allow obtaining a phenotype corresponding to the allele (G).

This nucleic acid of altered structure can consist of a regulatory polynucleotide (Pg) or a nucleic acid that encodes a CmWIP1 protein altered in its sequence.

Therefore, the object of the invention is a process for obtaining a transformed plant, belonging to the cucurbit family, bearing hermaphrodite flowers, characterized in that it comprises the following steps:

a) transformation of at least one plant cell of a plant of interest comprising an allele (G) by a regulatory polynucleotide (Pg) or by a nucleic acid encoding an altered CmWIP1 protein; or a recombinant vector comprising this nucleic acid;

b) selection of transformed cells obtained in step a) that have integrated into their genome at least one copy of a regulatory polynucleotide (Pg) or a nucleic acid encoding an altered CmWIP1 protein;

c) regeneration of a transformed plant from the transformed cells obtained in step b).

Without this forming part of the invention, the process may still comprise a stage d) of growth of plants obtained in stage c) to obtain one that no longer carries the (G) allele.

This type of procedure is particularly useful since it makes it possible to obtain plants that no longer comprise the (G) allele and that are hermaphroditic or gynoic type.

The above procedure can be carried out with the plant transformation procedure aimed at inserting the (A) allele which is described in PCT application No. WO 2007/125364.

This previous procedure for obtaining a transformed plant belonging to the cucurbitaceae family, which bears female flowers, is characterized in that it comprises the following stages:

a) transformation of at least one plant cell of a plant of interest that does not comprise the allele (A) in its genome, by a nucleotide sequence (NA); or a recombinant vector comprising this nucleic acid; b) selection of the transformed cells obtained in step a) that have integrated nucleic acid (NA) into their genome;

c) regeneration of a transformed plant from the transformed cells obtained in step b).

The invention also relates to a plant transformation process intended to replace allele (A) with allele (a).

The above procedure can be carried out with the plant transformation procedure for inserting allele (A) described in PCT application No. Wo 2007/125364.

This previous procedure for obtaining a transformed plant, belonging to the cucurbit family, that bears hermaphrodite flowers, is characterized in that it comprises the following stages:

a) the substitution in a plant of allele (A) by allele (a),

b) selection of the transformed cells obtained in step a) that have integrated the allele (A) into their genome, c) regeneration of a transformed plant from the transformed cells obtained in step b).

Without this forming part of the invention, the process can still comprise a stage d) of growth of plants obtained in stage c) to obtain one that no longer carries the allele (A).

Therefore, the above procedures can be combined among them merging in Table 1, so that exclusively female or exclusively hermaphroditic plants are obtained, whose industrial interest was previously exposed.

To simplify the procedures for obtaining an exclusively female or exclusively hermaphroditic transformed plant, it is possible to carry out a previous step in the previously defined procedures, in the course of which the mutations of the genes (A / a) and (G / g) naturally present in the plant are carried out, for example, by means of random insertion of the Mutator transposon in a population of wild phenotype plants, then detecting in the mutants obtained, those among these mutants that are genotype (aagg), for for example, by means of probes or nucleotide primers described in the examples.

According to this preferred embodiment, the transformed plant according to the invention is characterized in that it has a genotype (aagg) and has exclusively hermaphrodite flowers.

In one embodiment of the methods for obtaining a transformed plant described above, the polynucleotide (NG), when used, comprises an inducible activating regulatory polynucleotide (PG).

The plants obtained according to the above process exclusively bear hermaphroditic flowers and are therefore particularly interesting from an industrial point of view, since they are not capable of self-pollination.

Therefore, these plants can be used in pure line creation procedures.

Processes for transforming plants according to the invention.

In the context of this invention, the most widespread methods for introducing nucleic acids into plant cells can be used.

The transformation of plant cells can be carried out by various methods such as, for example, the transfer of the aforementioned vectors into plant protoplasts after an incubation of the latter in a polyethylene glycol solution in the presence of divalent divalent cations (Ca 2+) , electroporation (Fromm et al. 1985), the use of a particle gun, or cytoplasmic or nuclear microinjection (Neuhaus et al, 1987).

One of the plant cell transformation methods that can be used in the context of the invention is the infection of plant cells by a bacterial cell host that comprises the vector containing the sequence of interest. The cell host can be Agrobacterium tumefaciens (An et al. 1986), or A. rhizogenes (Guerche et al. 1987).

Preferably, the transformation of plant cells is carried out by transferring the T region of the extrachromozomal circular tumor-inducing plasmid Ti of A. tumefaciens, using a binary system (Watson et al., 1994). To do this, two vectors are constructed. In one of these vectors, the T-DNA region was deleted by deletion, with the exception of the left and right edges, a marker gene being inserted between them to allow selection of plant cells. The other member of the binary system is a Ti-helper plasmid, a modified plasmid that no longer has T-DNA but always contains the vir virulence genes necessary for the transformation of the plant cell. This plasmid is maintained in Agrobacterium.

According to a preferred mode, the method described by Ishida et al. (1996) can be applied for the transformation of dicotyledons. According to another protocol, the transformation is carried out according to the method described by Finer et al. (1992) using a tungsten or gold particle gun.

The present invention is also illustrated, but not limited to, in the following examples.

Examples

Example 1: Identification of the genetic control element (G / g)

Mapping-based g-site cloning is performed by selection of recombination elements in a population of 12,660 plants that secrete the g allele. An F1 generation plant derived from the growth of PI124112 (AG- monoecious genotype) x Gynadou (gynoic genotypes A-gg) was backcrossed with Gynadou in order to generate an F2 generation population, for analysis purposes. The g site was located in an interval between markers M261 and M365 on chromosome 4 (see Figure 1A).

Markers M261 and M365 were anchored in four different BAC clones from a monoecious genotype genomic library, in order to generate a physical map of the site.

An analysis of the SNPs in this range showed two highly critical recombinants, ultimately allowing the g-site to be reduced to a 1.4 kb region in the monoecious BAC 102 (see Figure 1C). The annotation of this clone allowed the identification of 8 open reading frames (ORFs) in this region (see Figure 1B).

No significant polymorphism was found in the 8 open reading frames that surround this interval, when the corresponding region was sequenced in the gynoid genotype that carries the recessive allele.

However, an 8 kb insert was found within the 1.4 kb region delimited by positional cloning (Figure 1B). This insert corresponds to the hAT family DNA transposon , called Gyno-hAT (Sequence No. 14), a very extended group of transportable elements (TEs).

Because this DNA transposon was found by positional cloning in the shortest interval, and because this transposon corresponds to the only significant polymorphism between the monoecious and gynoic genotypes at this location, this transportable element (TE) has a very high probability of being responsible for the sex determination phenotype.

Example 2: characterization of the genetic control element (G / g)

Because transportable elements (TEs) are subject to epigenetic inhibition ("silencing" that induces overexpression of transposition and illegitimate genomic clustering, the methylation status of the transposon DNA at the g site was examined by PCR amplification. sensitive to McrBC endonuclease The transposon was found to be highly methylated (see Figure 2).

The potential extent of DNA methylation of the g-site environment genes was analyzed by performing an McrBC endonuclease-sensitive PCR amplification on the three open reading frames (ORFs) closest to the transposon.

This analysis showed that the single ORF3 reference open reading frame that is located less than 1 kb away from the g-site was specifically methylated in the transposon-bearing gynoid genotype (Figure 3A). The ORF3 open reading frame encodes a zinc finger transcription factor C2H2 and is homologous to members of the plant-specific WIP subfamily (Sagasser et al., 2002).

In order to obtain more precise information on the DNA methylation profiles of the CmWIP1 genomic sequence, the entire CmWlP1 gene was selected by quantitative PCR sensitive to the endonuclease McrBC in the plants PI124112 (G-) and Gynadou (gg). (see figure 3B). Gynadou genomic DNA was shown to be highly methylated in the promoter region in the vicinity of the transcriptional start site, compared to the monoecious genotype (Figure 3B).

DNA methylation was also highest in the first exon, the intron, and in the vicinity of the 3 'UTR sequence of the genoic genotype, but less so.

In order to continue the study of the DNA methylation profiles of CmWIP1, bisulfite sequencing was performed on the highly methylated region of the CmWIP1 promoter. Bisulfite sequencing conformed the hypermethylation phenotype of gynoean plants.

It should be understood that methylation was always higher in the promoter of plants bearing the transposon to the g-site and methylation was also observed in cytosine residues that were completely unmethylated in the monoecious genotype (see Figure 3C).

Interestingly, however, significant DNA methylation was noted at the PI124112 promoter. The fact that significant DNA methylation was detected at the CmWIP1 promoter could explain the considerable extent of expression inhibition ("silenced") when a transportable element (TE) was inserted in the vicinity. The hypermethylation of CmWIP1 was confirmed in the different recessive genetic backgrounds for the g allele, which reinforces the correlation between the presence of the transposon DNA and the highest methylation status of this gene (Figure 4).

These results strongly suggest that hypermethylation of CmWIPI, which is due to an extension of inhibition due to the transposon ("silenced"), could be the cause of sex determination in plants that carry the g allele.

Excessive methylation of cytosine residues in the promoter region was associated with reduced gene expression, which could lead to decreased levels of CmWIP1 transcript production.

Example 3: control element expression regulation profiles (G / g)

In order to test the hypothesis according to which hypermethylation of the promoter region of the CmWIP1 gene could induce a decrease in the expression of this gene, the regulation profiles of CmWIP1 mRNA were analyzed. The expression levels of CmWIP1 were determined by quantitative PCR during sex determination and flower development.

The relative levels of transcripts were compared in male PI124112 (G-) flowers and in female Gynadou (gg) flowers.

In general, the determination of sex and flower development in cucurbits were divided into 12 phases, which go from the beginning of the flower meristem in anthesis (Bai et al., 2004). Sex determination procedures, that is, the arrest of the inappropriate sex organ in the initial bisexual flower bud, takes place approximately in stages 7 and 8.

In the present case, it has been shown that the CmWIP1 gene was strongly expressed in male flower buds in phase 6 and that its expression decreased rapidly in later stages (Figure 5). In contrast, in female flower buds, CmWIP1 mRNA was detected at a very low level, whatever the stage of flower development. Notably, the high and transient expression of CmWIP1 in male flower buds in an early stage of reproductive development coincides with the arrest of carpel development described in recent work on cucurbits (Hao et al., 2003; Bai et al. al., 2004).

In order to determine the space-time expression profile of CmWIP1 mRNA, in situ hybridizations of male PI142112 (G-) flowers and female Gynadou (gg) flowers were performed. CmWIP1 mRNA was detected early during flower development around phase 6 in male flower buds, which is consistent with the results of quantitative PCR analysis.

The location of CmWIP1 mRNA was found to be strongly confined to the fourth spiral of male flower buds. This location corresponds to the primary carpel that will stop in its development in the immediate later phase, in the male flowers.

CmWIP1 mRNA was not detectable in female flower buds at any stage of development. These results confirm the greatly reduced expression that was already shown by quantitative PCT analysis in male Gynadou flowers (see Figure 7).

CmWIP1 expression levels were also very low during the development phases associated with sex determination (up to phase 6) in hermaphroditic flower buds (aagg) and in female flower buds of monoecious genotype (Figure 6). In other words, the expression of CmWIP1 in the early phase of flower development was always low in flowers that developed into a mature female structure in all genotypes. The set of these results strongly suggests that CmWIP1 has a role in sex determination in melon, particularly in the arrest of carpel development in male flower buds.

Example 4: determination of the flower type under the control of the element (G / g)

In order to deepen the study of the role of CmWIP1 in determining sex and floral development in melon, a reverse genetics proposal was developed in order to identify the function of this protein.

A strategy that uses the TILLING technique (for Targeted Induced Local-scale Lesions in Genome ”) was used in order to select a population of monoecious genotype (A-G-) mutagenized with ethylmethanesulfonate (EMS). The choice of the population focused on the two exons of CmWIP1. Based on alignment with close homologs, the protein, CmWIP1 is composed of two different domains, respectively, a specific N-terminal domain and a conserved C-terminal domain in zinc finger WIP proteins.

Three mutants were identified in the sequence encoding CmWIP1 that have a single base substitution. Nucleotide substitutions result in the following amino acid modifications: L77F, P193L, and S306F. The amino acid substitutions were located either in the specific N-terminal part of CmWIP1 or in the highly conserved C-terminal domain of the protein.

Floral phenotypes were observed in M2 homozygous mimic plants, compared to wild-type homozygous plants of the same EMS family.

The three mimicking alleles showed new carpel development, compared to wild-type male chlorines (Figure 7).

Mutants P193L and S306F became completely gynoic, confirming that CmWIPI is the gene for gynoesia. The L77F mutant is a weak mutant.

The results of the examples show that the CmWIPI epilele that was found in genoic plants is clearly transmissible and co-segregates with the phenotype, since genotype-phenotype relationships were observed in different genetic backgrounds (see Figure 4) and in 12 thousand gametes through positional cloning experiments. Interestingly, and consistent with epigenetic regulation, reversal phenotypes were occasionally observed during somatic development, which were correlated with demethylation of the CmWIP1 DNA sequence.

The results of the examples, which demonstrate the implication of the regulation of the CmWIP1 gene in the determination of floral sex, are all the more interesting since the genes that encode proteins in WIP-type zinc fingers are found in a wide variety of plants. including monocots, gymnosperms and mosses. Furthermore, it is recalled that the conservation of the C-terminal domains of proteins in WIP-type zinc fingers is very high.

Consequently, carrying out a control of the regulation of CmWIP1, or of genes of the same family in genomes of dicotyledonous, monocotyledonous, gymnosperm and moss plants, at the transcriptional or posttranscriptional level during early floral development, is of a nature that orients of controlled form the development of floral type in plants.

Example 5: spatial and temporal expression of the genetic control element (A / a)

To study the expression of the control genetic element A / a in the form of the A allele, in situ hybridizations were carried out using specific probes of the A allele on plants and, more precisely, on floral meristems of male, female and hermaphrodite plants, of genotype AA GG, aa GG, AA gg and aa gg. In floral meristems A, the expression is locally elevated and the hybridization signal is detected specifically in the primordia of the carpels of female flowers and hermaphrodites of monoecious, andromonoic, gynoecious and hermaphrodite plants. Referring to the different development phases of the flower described for cucumber (Bai et al., 2004), it seems that in melon the gene (A / a) is expressed in an early phase of the development of floral meristems before that a morphological distinction can be made between male flowers and female flowers.

In male and hermaphroditic flowers, no expression was detected in anthers. These data indicate that the expression of the A allele in the carpels of female flowers prevents the development of stamens. Because, in hermaphroditic flowers, the recessive allele (a) presents the same expression profile as allele A in female flowers, it can be concluded that the function of gene A depends on its tissue expression specificity, as well as on the nature of the synthesized ACS protein.

Example 6: transgenesis in Arabidopsis thaliana

The potential effects of the A / a gene and the ACS protein on the floral sexual phenotype and flower architecture in non-cucurbit plants were studied by transformation of Arabidopsis thaliana by Agrobacterium. Transgenic Arabidopsis plants that carry the melon A or allele present a phenotype at the level of floral architecture and siliques (Figures 9a and 9B). Indeed, the siliques of Arabidopsis transformants are shorter than those of a wild Arabidopsis plant and the architecture of the transforming Arabidopsis flowers is greatly affected. These results make it possible to extend the use of the melon gene (A / a) to dicotyledonous plants not belonging to the cucurbit family.

In summary, thanks to the cloning of gene A (called CmACS-7) and gene G (called CmWIP1), it has been shown that the expression of the CmWIP1 protein in the carpel inhibits the development of the carpel and the expression of CmACS-7 o any other enzyme capable of producing ethylene in the carpel, inhibits the development of stamens. Therefore, a person skilled in the art can express the CmWIP1 protein in the carpel of a plant, preferably a cucurbit, to block the development of the carpel or, conversely, inhibit the expression of the CmWIP1 protein in the carpel to promote development. of the carpel.

One skilled in the art can also express the CmACS-7 protein in the carpel of a plant, preferably a cucurbit to block the development of the stamens or, on the contrary, inhibit the expression of the CmWIP1 protein in the carpel to promote development. of the stamens. Therefore, the combination of the expression of the active or mutant proteins of CmACS-7 and CmWIP1 will allow the generation of different floral types. described in table 1.

The authors have also identified the orthologs of CmACS-7 and CmWIPI in other species. In cucumber ( Cucumis sativa) place M encodes the ortholog of SEQ ID N °

Sequence table

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Claims (21)

1. Use of a combination of two genetic elements to control the development of the floral type of a dicotyledonous plant, said combination comprising, respectively: a) a first genetic control element (A / a) present in said dicotyledonous plant, in the form of a dominant allele (A) and a recessive allele (a), in which: - the dominant allele (A) consists of a nucleic acid (NA) that allows the expression of the ACS protein (aminocyclopropane-carboxylate synthase) of sequence SEQ ID N ° 3 - the recessive allele (a) is distinguished from the dominant allele in that said recessive allele (a) is non-functional in said dicotyledonous plant, and b) a second genetic control element (G / g) present in said dicotyledonous plant, in the form of a dominant allele (G) and a recessive allele (g), in which: - the dominant allele (G) consists of a nucleic acid (NG) that allows the expression of the CmWIPI protein ("C. melo Zinc Finger Protein") of sequence SEQ ID N ° 12, - the recessive allele (g) is distinguished from the dominant allele in that said recessive allele (g) is non-functional in said dicotyledonous plant, it being understood that at least the second genetic control element has been artificially introduced into said dicotyledonous plant. 2. Use of a combination of two genetic elements according to claim 1, characterized in that: - the dominant allele (A) consists of a nucleic acid (NA) comprising: (i) a regulatory polynucleotide (PA) functional in a dicotyledonous plant, and (ii) a nucleic acid whose expression is regulated by the regulatory polynucleotide (PA), said nucleic acid encoding the ACS protein (aminocyclopropane carboxylate synthase) of sequence SEQ ID N ° 3, - the non-functional recessive allele (a) is distinguished from the dominant allele by a non-functional nucleic acid and comprises: (i) a regulatory polynucleotide (Pa) whose nucleotide sequence comprises an insertion, a substitution or a deletion with respect to the nucleic acid ranging from nucleotide 1 to nucleotide 5907 of the sequence SEQ ID No. 1 and leading to a reduced or no expression of the ACS protein of sequence SEQ ID No. 3, (ii) a nucleic acid (Na) that encodes a protein that differs from the ACS protein of sequence SEQ ID N ° 3 by the substitution, insertion or deletion of one or more amino acids and that does not possess the biological activity of said protein ACS in said dicotyledonous plant, and - the dominant allele (G) consists of a nucleic acid (NG) comprising: (i) a regulatory polynucleotide (PG) functional in a dicotyledonous plant and (ii) a nucleic acid whose expression is regulated by the regulatory polynucleotide (PG), in which said nucleic acid encodes the CmWIP1 protein ("C. melo Zinc Finger Protein") of sequence SEQ ID N ° 12, the non-functional recessive allele (g) is distinguished from the dominant allele (G) by a non-functional nucleic acid and comprises: (i) a regulatory polynucleotide (Pg) whose nucleotide sequence comprises at least one mutation, an insertion or a deletion with respect to the nucleic acid of sequence SEQ ID N ° 13 leading said nucleic acid to a reduced or null expression of the CmWIP1 protein of sequence SEQ ID No. 12, or (ii) a nucleic acid (Ng) that encodes a protein that differs from the CmWIP1 protein of sequence SEQ ID N ° 12 by the substitution, insertion or deletion of one or more amino acids and that does not possess the biological activity of said protein CmWIP1 in said dicotyledonous plant. 3. Use of a combination of two genetic elements according to one of claims 1 and 2, characterized in that the nucleic acid encoding the CmWIP1 protein comprises, from the 5 'end to the 3' end, at least: (i) a sequence that has at least 95% identity with the polynucleotide ranging from nucleotide 3000 to nucleotide 3617 of SEQ ID No. 10, and (ii) a sequence that has at least 95% identity with the polynucleotide ranging from nucleotide 5458 to nucleotide 5901 of SEQ ID No. 10. 4. Use of a combination of two genetic elements according to one of claims 2 and 3, characterized in that the regulatory polynucleotide (PG) comprises the nucleotide sequence SEQ ID No. 11. 5. Nucleic acid comprising, from the 5 'end to the 3' end, at least: (i) a sequence that has at least 98.5% identity with the polynucleotide ranging from nucleotide 3000 to nucleotide 3617 of the sequence SEQ ID No. 10, and (ii) a sequence that has at least 99.5% identity with the polynucleotide ranging from nucleotide 5458 to nucleotide 5901 of the sequence SEQ ID No. 10. 6. Nucleic acid in allele form (G) as defined in claim 5, of sequence SEQ ID No. 10. 7. Nucleic acid comprising a nucleotide sequence ranging from nucleotide 3000 to nucleotide 5901 of the sequence SEQ ID No. 10. 8. Nucleic acid, which comprises a nucleotide sequence that carries at least one alteration chosen from a mutation, an insertion or a deletion, with respect to the nucleic acid that goes from nucleotide 1, to nucleotide 2999 of the sequence SEQ ID N 10, leading said altered nucleic acid to an altered expression of the CmWIP1 protein of the sequence SEQ ID N ° 12, when controlling the expression of said protein, with respect to the expression of the CmWIP1 protein controlled by the nucleic acid ranging from nucleotide 1 to nucleotide 2999 of the sequence SEQ ID No. 10. 9. Recombinant vector comprising a nucleic acid, intended to be artificially introduced into a dicotyledonous plant, in which the nucleic acid is chosen from: a) a genetic control element (G / g), where the control genetic element (G / g) occurs as a dominant allele (G) or as a recessive allele (g): - the dominant allele (G) consisting of a nucleic acid (NG), comprising: (i) a regulatory polynucleotide (PG) functional in a dicotyledonous plant, and (ii) a nucleic acid whose expression is regulated by the regulatory polynucleotide (PG), said nucleic acid encoding the expression of the CmWIP1 protein ("C. melo Zinc Finger Protein") of sequence SEQ ID N ° 12, - the non-functional recessive allele (g) that is distinguished from the dominant allele (G) in a non-functional nucleic acid and comprising: (i) a regulatory polynucleotide (Pg) whose nucleotide sequence comprises at least one mutation, an insertion or a deletion with respect to the nucleic acid of sequence SEQ ID N ° 13, said nucleic acid leading to a reduced or null expression of the protein CmWIP1 of sequence SEQ ID No. 12, or (ii) a nucleic acid (Ng) that encodes a protein that differs from the CmWIP1 protein of sequence SEQ ID N ° 12 in the substitution, insertion or deletion of one or more amino acids and that does not possess the biological activity of said protein CmWIP1 in said dicotyledonous plant, or b) a nucleic acid according to any one of claims 5 to 8. 10. Host cell transformed by a recombinant vector according to claim 9, wherein the host cell comprises, in its genome, a nucleic acid artificially introduced by said recombinant vector, wherein the nucleic acid is chosen from: a) a genetic control element (G / g), where the control genetic element (G / g) occurs as a dominant allele (G) or as a recessive allele (g): - the dominant allele (G) consisting of a nucleic acid (NG), comprising: (i) a regulatory polynucleotide (PG) functional in a dicotyledonous plant, and (ii) a nucleic acid whose expression is regulated by the regulatory polynucleotide (PG), said nucleic acid encoding the expression of the protein CmWIPI ("C. melo Zinc Finger Protein") of sequence SEQ ID N ° 12, - the non-functional recessive allele (g) that is distinguished from the dominant allele (G) in a non-functional nucleic acid and comprising: (i) a regulatory polynucleotide (Pg) whose nucleotide sequence comprises at least one mutation, an insertion or a deletion with respect to the nucleic acid of sequence SEQ ID N ° 13, said nucleic acid leading to a reduced or null expression of the protein CmWIP1 of sequence SEQ ID No. 12, or (ii) a nucleic acid (Ng) that encodes a protein that differs from the CmWIP1 protein of sequence SEQ ID N ° 12 in the substitution, insertion or deletion of one or more amino acids and that does not possess the biological activity of said protein CmWIP1 in said dicotyledonous plant, or b) a nucleic acid according to any one of claims 5 to 8. Host cell according to claim 10, characterized in that it is a plant cell belonging to the cucurbit family and, preferably, to the species Cucumis melo. 12. Plant belonging to the cucurbit family transformed by means of a recombinant vector according to claim 9, in which said plant comprises, in its genome, a nucleic acid artificially introduced by said recombinant vector, in which the nucleic acid is chosen from: a) a genetic control element (G / g), where the control genetic element (G / g) occurs as a dominant allele (G) or as a recessive allele (g): - the dominant allele (G) consisting of a nucleic acid (NG), comprising: (i) a regulatory polynucleotide (PG) functional in a dicotyledonous plant, and (ii) a nucleic acid whose expression is regulated by the regulatory polynucleotide (PG), said nucleic acid encoding the expression of the CmWIP1 protein ("C. melo Zinc Finger Protein") of sequence SEQ ID N ° 12, - the non-functional recessive allele (g) that is distinguished from the dominant allele (G) in a non-functional nucleic acid and comprising: (i) a regulatory polynucleotide (Pg) whose nucleotide sequence comprises at least one mutation, an insertion or a deletion with respect to the nucleic acid of sequence SEQ ID N ° 13, said nucleic acid leading to a reduced or null expression of the protein CmWIP1 of sequence SEQ ID No. 12, or (ii) a nucleic acid (Ng) that encodes a protein that differs from the CmWIP1 protein of sequence SEQ ID N ° 12 in the substitution, insertion or deletion of one or more amino acids and that does not possess the biological activity of said protein CmWIP1 in said dicotyledonous plant, or b) a nucleic acid according to any one of claims 5 to 8. 13. A transformed plant according to claim 12, characterized in that it comprises at least one allele (G) as defined in claim 1. 14. Host cell, transformed by: (i) a recombinant vector comprising a nucleic acid chosen from: - a nucleic acid that determines an allele A or a as defined in claim 1, - a nucleic acid comprising, from the 5 'end to the 3' end, at least: (i) a sequence that has at least 95% identity with the polynucleotide ranging from nucleotide 5907 to nucleotide 6086 of the sequence SEQ ID N ° 1, (ii) a sequence that has at least 95% identity with the polynucleotide ranging from nucleotide 6181 to nucleotide 6467 of the sequence SEQ ID No. 1, and (iii) a sequence that has at least 95% identity with the polynucleotide ranging from nucleotide 7046 to nucleotide 7915 in the sequence SEQ ID N ° 1, - a nucleic acid in the form of allele (A), as defined in claim 2, of sequence SEQ ID N ° 1; - a nucleic acid in the form of allele (a), as defined in claim 2, of sequence SEQ ID N ° 2, - a nucleic acid comprising a sequence of nucleotides ranging from nucleotide 1 to nucleotide 5906 of the sequence SEQ ID N ° 1, - a nucleic acid comprising a nucleotide sequence that carries at least one alteration chosen from a mutation, an insertion or a deletion, with respect to the nucleic acid that goes from nucleotide 1 to nucleotide 5907 of the sequence SEQ ID N ° 1 , leading said altered nucleic acid to an altered expression of the ACS protein of sequence SEQ ID N ° 3, when controlling the expression of said protein, with respect to the expression of the ACS protein controlled by the nucleic acid that goes from nucleotide 1 up to nucleotide 5907 of the sequence SEQ ID N ° 1, and - a nucleic acid comprising a sequence ranging from nucleotide 1 to nucleotide 3650 of the sequence SEQ ID N ° 2, and (ii) a recombinant vector according to claim 9, wherein the host cell comprises, in its genome, a nucleic acid artificially introduced by said recombinant vector. Host cell according to claim 14, characterized in that it is a plant cell belonging to the cucurbit family and, preferably, to the spice cucumis melo. 16. Nucleic acid usable as a primer, which specifically hybridizes with a nucleic acid according to any one of claims 5 to 8. 17. Method for detecting the presence of a (G) or (g) allele as defined in claim 1, said method comprising the following steps: 1) contacting a probe or a plurality of nucleotide probes that specifically hybridize with a nucleic acid according to any one of claims 5 to 8, with the sample to be tested, 2) detect the complex eventually formed between the probe (s) and the nucleic acid present in the sample. 18. Procedure for obtaining artificially mutated plants in the gene encoding the CmWIP1 protein, which comprises the following steps: a) generate a collection of mutant dicotyledonous plants by chemical mutagenesis. b) selecting from the collection of mutant plants generated in step a) the plants that have one or more than one mutation in the gene encoding the CmWIP1 protein, c) selecting among the mutant plants selected in step b), the plants that express the phenotype associated with allele (g) of the genetic element (G / g) as defined in claim 1. 19. The method according to claim 18, characterized in that the plants are also mutated in the gene encoding the ACS protein and in that: - plants are selected in step b) (i) that have one or more than one mutation in the gene encoding the CmWIP1 protein and (ii) that have one or more than one mutation in the gene that encodes the protein ACS, - in step c), among the plants mutated in the gene encoding the CmWIP1 protein and in the gene encoding the ACS protein, the plants that express at the same time the phenotype associated with allele (g) of the genetic element ( G / g) and the phenotype associated with allele (a) of the genetic element (A / a). 20. Procedure for obtaining a transformed plant belonging to the Cucurbitaceae family, characterized in that it comprises the following stages: a) transformation of at least one plant cell of a plant of interest that does not comprise the allele (G) as defined in claim 1 in its genome, by means of a nucleotide sequence (NG) as defined in claim 1, or a recombinant vector comprising this nucleic acid; b) selection of the transformed cells obtained in step (a) that have integrated into their nucleic acid (NG); c) regeneration of a transformed plant from the transformed cells obtained in step b). 21. Procedure for obtaining a transformed plant belonging to the cucurbit family, characterized in that it comprises the following stages: a) transformation of at least one plant cell of a plant of interest comprising the allele (G) as defined in claim 1 in its genome, by a non-functional regulatory polynucleotide (Pg) or by a nucleic acid (Ng) that encodes an altered CmWIP1 protein, as defined in claim 1, or by an antisense nucleic acid (NG), b) selection of transformed cells from a plant obtained in step a) and that have integrated into their genome at least one copy of a non-functional regulatory polynucleotide (Pg) or of a nucleic acid (Ng) that encodes a CmWIP1 protein altered, as defined in claim 1, or no longer comprising the allele (G), c) regeneration of a transformed plant from the transformed cells obtained in step b).